Biologically active peptides

ABSTRACT

Thirty substantially pure and biologically active peptides are disclosed. Nucleic acids that have sequences coding for the biologically active peptides and pharmaceutical formulations produced thereform are also disclosed.

RELATED APPLICATIONS

This application is a divisional of copending U.S. patent applicationSer. No. 10/237,405, filed Sep. 5, 2002, which is a continuation-in-partof U.S. patent application Ser. No. 10/178,684, filed Jun. 20, 2002(abandoned), which is a continuation-in-part of U.S. patent applicationSer. No. 09/904,492, filed Jul. 13, 2001 (abandoned). The disclosures ofall of the above-referenced priority documents are incorporated byreference herein in their entireties.

FIELD OF INVENTION

The present invention is related to the field of immunology. Inparticular, the present invention is directed to peptides and theirpharmaceutical compositions which are capable of modulating immuneresponses.

BACKGROUND OF INVENTION

Spleen extracts from mammals have been used successfully for years asmedical immune modulators. However, the molecular composition of suchextracts is unknown and therefore the exact pharmacological actions ofthese extracts cannot be identified. The dosage response function alsocannot be accurately titrated because of the intrinsic heterogeneousnature of these extracts. Because of such heterogeneity, activeingredients in spleen extracts cannot be singled out and theirpharmacological actions cannot be fully utilized. And, no matter howcareful the extraction process can be, there is always the possibilityof transmission of diseases of animal origin to human if animal tissueis used as the raw material for extraction.

U.S. Pat. No. 3,992,364 to Kuhlmey describes a physiologically activepolypeptide produced from an animal spleen extract. The exact sequenceof the peptide was not disclosed and the preparation was described asbeing useful for decreasing cholesterol levels and increasing the 17keto-steroid elimination of human patients.

SUMMARY OF INVENTION

It is an object of the present invention to identify biologically activepolypeptides.

A study was conducted in which a number of peptides have been identifiedfrom a porcine spleen extract according to the method described in U.S.Pat. No. 3,992,364. The active fraction is also sold under the tradename “Polyerga”. The peptides in the extract were separated bypreparative HPLC followed by analysis using electrospray ionisation massspectroscopy (ESMS) and matrix assisted laser desorption ionisation massspectroscopy (MALDI-MS) at the W. M. Keck Foundation BiotechnologyResource Laboratory at Yale University. Once the identity of thepeptides was found, they were individually synthesized by standardchemical methods. Using known animal and in vitro methods, theimmunological function of these peptides was analyzed. The in vivo studyof the effect of the peptides on immunity was performed using methodsdescribed in the following references. The peptides are given codeshaving the letters CMS followed by a number. The peptide sequence andthe corresponding ID numbers are shown in Table A. A total of 30peptides have been identified as having in vivo biological activities.The effect of the peptides on kidney function, liver function, cancerand body weight was also analyzed. TABLE A Sequence Listing Peptide IDNo. Name Peptide Sequence 1 CMS001 Pro Thr Thr Lys Thr Tyr Phe Pro HisPhe 2 CMS002 Val Val Tyr Pro Trp Thr Gln Arg Phe 3 CMS008 Lys Ala ValGly His Leu Asp Asp Leu Pro Gly Ala Leu 4 CMS010 Val Ala Pro Glu Glu HisPro Thr Leu Leu Thr Glu Ala Pro Leu Asn Pro Lys 5 CMS012 Leu Gly Met GluAla Cys Gly Ile His Glu Thr Thr Tyr 6 CMS013 Leu Arg Val Ala Pro Glu GluHis Pro Val Leu 7 CMS014 Ala Ala His His Pro Asp Asp Phe Asn Pro Ser Val8 CMS015 Pro Ser Ile Val Gly Arg Pro Arg His Gln Gly Val Met 9 CMS016Ile Gly Met Glu Ser Ala Gly Ile His Glu Thr Thr Tyr 10 CMS018 Val GlyMet Gly Glu Lys Asp Ser Tyr 11 CMS019 Val Gly Met Gly Gln Lys Asp SerTyr 12 CMS020 Val Gly Met Gly Gln Lys Asp Ser Tyr Val 13 CMS021 Met AlaThr Ala Ala Ser Ser Ser Ser Leu 14 CMS022 Tyr Ser Phe 15 CMS023 Ala AlaPhe 16 CMS024 Tyr Ser Leu 17 CMS026 Thr Thr Tyr Asn Ser Ile Met 18CMS027 Phe Glu Glu Asn Met 19 CMS028 Phe Glu Pro Ser Phe 20 CMS029 PheAsn Glu Glu 21 CMS030 Phe Glu Glu Met 22 CMS032 Phe Glu Glu Glu 23CMS033 Phe Glu Ser Phe 24 CMS034 Pro Glu Asn Phe 25 CMS035 Phe Val AsnAsp 26 CMS036 Phe Gln Pro Ser Phe 27 CMS003 Phe Asn Phe Val Pro Pro 28CMS007 Ala Gly Asp Asp Ala Pro Arg Ala Val Phe 29 CMS009 Leu Arg Val AlaPro Glu Glu His Pro Thr Leu 30 CMS011 Arg Val Ala Pro Glu Glu His ProThr Leu

Accordingly, one aspect of the present invention relates tosubstantially pure peptides having sequences identified as sequence IDNo. 1 to sequence ID No.30. Thus the present invention also relates to asubstantially pure peptide comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs. 1-30. It also relates to asubstantially pure peptide consisting essentially of an amino acidsequence selected from the group consisting of SEQ ID NOs. 1-30. Theinvention also relates to a substantially pure peptide consisting of anamino acid sequence selected from the group consisting of SEQ ID NOs.1-30. In a specific embodiment, the peptides can modulate, but notlimited to modulating, one or more of the following: immune activity;hepatitis infection, including but not limited to hepatitis B infection;nephritis; the growth of a cancer, including but not limited to sarcoma,liver cancer, leukemia and melanoma; and body weight.

Another aspect of the present invention relates to substantially purepeptides that are functional derivatives of peptides having sequencesidentified as sequence ID No. 1 to 30. Thus the present inventionrelates also to a substantially pure peptide comprising an amino acidsequence which is a functional derivative of a biologically activepeptide, this biologically active peptide having an amino acid sequenceselected from the group consisting of SEQ ID NOs. 1-30. It also relatesto a substantially pure peptide consisting essentially of an amino acidsequence which is a functional derivative of a biologically activepeptide, this biologically active peptide having an amino acid sequenceselected from the group consisting of SEQ ID NOs. 1-30. The inventionalso relates to a substantially pure peptide consisting of an amino acidsequence which is a functional derivative of a biologically activepeptide, this biologically active peptide having an amino acid sequenceselected from the group consisting of SEQ ID NOs. 1-30. In a specificembodiment, the peptides that are functional derivatives can modulate,but not limited to modulating, one or more of the following: immuneactivity; hepatitis infection, including but not limited to hepatitis Binfection; nephritis; the growth of a cancer, including but not limitedto sarcoma, liver cancer, leukemia and melanoma; and body weight.

Another aspect of the present invention relates to nucleic acids thathave sequences coding for the peptides identified above as sequence IDNo.1 to 30. A further aspect of the present invention relates toexpression vectors that contain the nucleic acid sequences of thepeptides shown below as sequence ID No. 1 to 30. Thus, this aspect ofthe present invention also relates to a genetic vector comprising anucleotide sequence encoding a peptide comprising an amino acid sequenceselected from the group comprising of SEQ ID NOs. 1-30. It also relatesto a genetic vector comprising a nucleotide sequence encoding a peptideconsisting essentially of an amino acid sequence selected from the groupconsisting of SEQ ID NOs. 1-30. The invention also relates to a geneticvector comprising a nucleotide sequence encoding a peptide comprising afunctional derivative of a biologically active amino acid sequenceselected from the group consisting of SEQ ID NOs. 1-30. It also relatesto a genetic vector comprising a nucleotide sequence encoding a peptideconsisting essentially of a functional amino acid sequence which is afunctional derivative of a biologically active amino acid sequenceselected from the group consisting of SEQ ID NOs. 1-30.

Yet another aspect of the present invention relates to hybrid peptidescontaining a leader peptide adjacent a peptide, the peptide comprisingan amino acid sequence selected from the group consisting of SEQ ID NOs.1-30. The present invention also relates to hybrid peptides containing aleader peptide adjacent a peptide, the peptide comprising a functionalderivative of a biologically active peptide, this biologically activepeptide having an amino acid sequence selected from the group consistingof SEQ ID NOs. 1-30.

The present invention also relates a genetic vector comprising anucleotide sequence encoding a peptide comprising a leader amino acidsequence adjacent a peptide comprising a functional amino acid sequencewhich is a functional derivative of a biologically active amino acidsequence selected from the group consisting of SEQ ID NOs. 1-30. It alsorelates to a genetic vector comprising a nucleotide sequence encoding apeptide comprising a leader amino acid sequence adjacent a peptideconsisting essentially of a functional amino acid sequence which is afunctional derivative of a biologically active amino acid sequenceselected from the group consisting of SEQ ID NOs. 1-30.

In a specific embodiment, the peptides produced in any of theabove-described genetic vectors can modulate, but not limited tomodulating, one or more of the following: immune activity; hepatitisinfection, including but not limited to hepatitis B infection;nephritis; the growth of a cancer, including but not limited to sarcoma,liver cancer, leukemia and melanoma; and body weight.

Yet another aspect of the present invention relates to a micro-organismwith a genome comprising a nucleotide sequence encoding a peptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs. 1-30. It also relates to a micro-organism with a genomecomprising a nucleotide sequence encoding a peptide consistingessentially of an amino acid sequence selected from the group consistingof SEQ ID NOs. 1-30.

Yet another aspect of the present invention relates to a micro-organismwith genetic material comprising a nucleotide sequence encoding apeptide, preferably an exogenous peptide comprising a functional aminoacid sequence which is a functional derivative of a biologically activeamino acid sequence selected from the group consisting of SEQ ED NOs.1-30. It also relates to a micro-organism with a genetic compositioncomprising a nucleotide sequence encoding an exogenous peptideconsisting essentially of a functional amino acid sequence which is afunctional derivative of a biologically active amino acid sequenceselected from the group consisting of SEQ ID NOs. 1-30. Exogenouspeptide as used herein refers to a peptide having an amino acid sequencethat is different from any other peptides normally expressed by themicro-organism in its natural, unmodified form.

Yet another aspect of the present invention relates to a micro-organismwith a genetic composition comprising a nucleotide sequence encoding apeptide, preferably an exogenous hybrid peptide comprising a leaderamino acid sequence adjacent a peptide, the peptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs. 1-30. Italso relates to a micro-organism with a genome comprising a nucleotidesequence encoding a hybrid peptide comprising a leader amino acidsequence adjacent a peptide consisting essentially of an amino acidsequence selected from the group consisting of SEQ ID NOs. 1-30.

Yet another aspect of the present invention relates to a micro-organismwith a genetic composition comprising a nucleotide sequence encoding anexogenous hybrid peptide comprising a leader amino acid sequenceadjacent a peptide, the peptide comprising a functional amino acidsequence which is a functional derivative of a biologically active aminoacid sequence selected from the group consisting of SEQ ID NOs. 1-30. Italso relates to a micro-organism with a genetic composition comprising anucleotide sequence encoding an exogenous hybrid peptide comprising aleader amino acid sequence adjacent a peptide consisting essentially ofa functional amino acid sequence which is a functional derivative of abiologically active amino acid sequence selected from the groupconsisting of SEQ ID NOs. 1-30.

In a specific embodiment, the peptides produced in any of theabove-described micro-organism can modulate, but not limited tomodulating, one or more of the following: immune activity; hepatitisinfection, including but not limited to hepatitis B infection;nephritis; the growth of a cancer, including but not limited to sarcoma,liver cancer, leukemia and melanoma; and body weight.

Yet another aspect of the present invention relates to a pharmaceuticalcomposition comprising a substantially pure peptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs. 1-30.The invention also relates to pharmaceutical composition comprising asubstantially pure peptide consisting essentially of an amino acidsequence selected from the group consisting of SEQ ID NOs. 1-30.

The present invention also relates to a pharmaceutical compositioncomprising a substantially pure peptide comprising a functionalderivative of a biologically active peptide, this biologically activepeptide having an amino acid sequence selected from the group consistingof SEQ ID NOs. 1-30. It also relates to a pharmaceutical compositioncomprising a substantially pure peptide consisting essentially of afunctional derivative of a biologically active peptide, thisbiologically active peptide having an amino acid sequence selected fromthe group consisting of SEQ ID NOs. 1-30. It further relates topharmaceutical composition comprising a substantially pure peptideconsisting of functional derivative of a biologically active peptide,this biologically active peptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs. 1-30.

In a specific embodiment, the peptides present in any of theabove-described pharmaceutical compositions can modulate, but notlimited to modulating, one or more of the following: immune activity;hepatitis infection, including but not limited to hepatitis B infection;nephritis; the growth of a cancer, including but not limited to sarcoma,liver cancer, leukemia and melanoma; and body weight.

Yet a further aspect of the present invention relates to a method ofmaking a pharmaceutical composition comprising providing a substantiallypure peptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs. 1-30; and mixing said substantially purepeptide with a pharmaceutically acceptable carrier. It also relates to amethod of making a pharmaceutical composition comprising providing asubstantially pure peptide consisting essentially of an amino acidsequence selected from the group consisting of SEQ ID NOs. 1-30.

Another aspect of the present invention is a method of making apharmaceutical composition comprising providing a substantially purepeptide comprising an amino acid sequence which is a functionalderivative of a biologically active peptide, this biologically activepeptide having an amino acid sequence selected from the group consistingof SEQ ID NOs. 1-30; and mixing said substantially pure peptide with apharmaceutically acceptable carrier.

It further relates to a method of making a pharmaceutical compositioncomprising providing a substantially pure peptide consisting essentiallyof an amino acid sequence which is a functional derivative of abiologically active peptide, this biologically active peptide having anamino acid sequence selected from the group consisting of SEQ ID NOs.1-30; and mixing the substantially pure peptide with a pharmaceuticallyacceptable carrier.

In connection with any of the above-described method, the peptide canmodulate, but not limited to modulating, one or more of the following:immune activity; hepatitis infection, including but not limited tohepatitis B infection; nephritis; the growth of a cancer, including butnot limited to sarcoma, liver cancer, leukemia and melanoma; and bodyweight.

Yet a further aspect of the present invention relates to a method oftreatment of a human comprising administering a pharmaceuticallyeffective dose of a substantially pure peptide comprising an amino acidsequence selected from the group consisting of SEQ ID NOs. 1-30 to ahuman. It also relates to a method of treatment of a human comprisingadministering a pharmaceutically effective dose of a substantially purepeptide comprising an amino acid sequence which is a functionalderivative of a biologically active peptide, this biologically activepeptide having an amino acid sequence selected from the group consistingof SEQ ID NOs. 1-30.

In a specific embodiment, the peptides used for the treatment of humandescribed above may be used to modulate, but not limited to modulating,one or more of the following human conditions: immune activity;hepatitis infection, including but not limited to hepatitis B infection;nephritis; the growth of a cancer, including but not limited to sarcoma,liver cancer, leukemia and melanoma; and body weight.

In connection with any of the above-described nucleic acid sequences,the peptides and/or hybrid peptides expressed from these nucleic acidsequences can modulate, but not limited to modulating, the following:immune activity; hepatitis infection, including but not limited tohepatitis B infection; nephritis; the growth of a cancer, including butnot limited to sarcoma, liver cancer, leukemia and melanoma; and bodyweight.

Another aspect of the present invention relates to the method oftreating diseases comprising administering a pharmaceutically effectivedose of a substantially pure peptide having sequence ID No.1 to sequenceID No.30. In a specific embodiment, the peptides so administered canmodulate, but not limited to modulating, the following: immune activity;hepatitis infection, including but not limited to hepatitis B infection;nephritis; the growth of a cancer, including but not limited to sarcoma,liver cancer, leukemia and melanoma; and body weight.

As described above, another embodiment of the present invention is apeptide or polypeptide consisting essentially of a peptide of thepresent invention. As used herein, the terminology “consistingessentially of” refers to a peptide or polypeptide which includes theamino acid sequence of the peptides of the present invention along withadditional amino acids at the carboxyl and/or amino terminal ends andwhich maintains the activity of the peptides of the present inventionprovided herein. Thus, as a non-limiting example, where the activity ofthe peptide of the present invention is to modulate immune activity, apeptide or polypeptide “consisting essentially of” the peptide of thepresent invention will possess the activity of modulating immuneactivity as provided herein with respect to that peptide and will notpossess any characteristics which materially reduces the ability of thepeptide or polypeptide to modulate immune activity or which constitutesa material change to the basic and novel characteristics of the peptideas a modulator of immune activity. Thus, in the foregoing example, afull length naturally occurring polypeptide which has a primary activityother than modulating immune activity and which contains the amino acidsequence of a peptide of the present invention somewhere therein wouldnot constitute a peptide or polypeptide “consisting essentially of” apeptide of the present invention. Likewise, in the foregoing example, agenetically engineered peptide or polypeptide which has a primaryactivity other than modulating immune activity but includes the aminoacid sequence of a peptide of the present invention somewhere thereinwould not constitute a peptide or polypeptide “consisting essentiallyof” a peptide of the present invention.

Besides the example of immune activity modulation used for illustrationabove, the foregoing definition also applies to all the peptides of thepresent invention with respect to the activities provided for suchpeptides. In particular, the foregoing definition applies to peptides ofthe invention having activities in modulating the extent of a viralinfection, modulating the extent of a hepatitis infection, modulatingthe extent of nephritis, modulating the growth of a cancer, ormodulating body weight as set forth in the detailed description below.

Those skilled in the art can readily determine whether a peptide orpolypeptide consists essentially of a peptide of the present inventionunder the foregoing definitions by measuring the activity of the peptideor polypeptide using the assays for modulation of immune activity,modulating the extent of a viral infection, modulating the extent of ahepatitis infection, modulating the extent of nephritis, modulating thegrowth of a cancer, or modulating body weight which are provided hereinwith respect to a particular peptide of the present invention.

In the preferred embodiment, the terminology “consisting essentially of”may also refer to peptides or polypeptides which have less than 20 aminoacid residues in addition to the peptide according to the presentinvention. In a more preferred embodiment, the same terminology refersto a peptides with less than 15 amino acid residues in addition to thepeptide according to the instant invention. In an even more preferredembodiment, the same terminology refers to a peptides with less than 10amino acid residues in addition to the peptide according to the instantinvention. In another preferred embodiment, the same terminology refersto peptides or polypeptides with less than 6 amino acids in addition toone of the peptide of the present invention. In another preferredembodiment, the same terminology refers to peptides or polypeptides withless than 4 amino acids in addition to one of the peptide of the presentinvention. In the most preferred embodiment, the same terminology refersto peptides or polypeptides with less than 2 amino acids in addition toone of the peptide of the present invention.

DETAILED DESCRIPTION

The peptides can be readily synthesized by standard synthetic methodsfrom L-amino acids, but may also be synthesized by genetic engineeringmethods using nucleic acids that have sequences encoding the individualpeptides.

I. Biological Activity

In order to investigate the possible biological activity of peptides,the immunological effect of the peptides on animal model was examined,with procedures compliant to the “Principles of Pre-clinical Research ofNew Drugs” issued by Ministry of Health of People's Republic ofChina^([1]).

The T lymphocyte transformation test, NK cell cytotoxicity activitytest, and the T lymphocyte IL-2 and IFN-γ secretion test were used todetect any possible effect of peptides on specific cellular immunefunction. The carbon particle clearance test was used to detect anypossible effect of peptides on non-specific cellular immune function.The Sheep Red Blood Cell (SRBC) hemolysis test was used to detect anypossible effect of peptides on humoral immune function. The immunityorgan weight test was used to detect any possible effect of peptides atthe organ level.

In this study, the saline group was used as negative control, while theIL-2 and IFN-γ groups were used as positive controls, since IL-2 andIFN-γ are well-studied immunostimulants^([)10]. Four arbitraryconcentrations of sample peptides were used in this study, to cover a1000 fold dosage range. Due to the intrinsic complexity of in vivoimmunological response, and the lack of prior knowledge on the dosageversus response function, therefore any statistically significantdifference over the negative control in any of the dosage groups hasbeen scored as positive biological activity.

Results of this study were as follows:

-   -   1. Peptides CMS001, CMS002, CMS003, CMS007, CMS008, CMS009,        CMS010, CMS011, CMS012, CMS015, CMS019, CMS021, CMS029, and        CMS034 were found to be able to enhance T lymphocyte        transformation, having statistically significant difference from        the saline normal control group. Peptides CMS014 and CMS036 were        also found to be able to inhibit T lymphocyte transformation,        having statistically significant difference from the saline        normal control group.    -   2. Peptides CMS001, CMS002, CMS003, CMS008, CMS009, CMS010,        CMS011, CMS012, CMS013, CMS015, CMS016, CMS020, CMS021, CMS022,        CMS023, CMS024, CMS026, CMS027, CMS028, CMS029, CMS030, CMS032,        CMS033, CMS034, CMS035, and CMS036 were found to be able to        increase the cytotoxic activity of NK cells, having        statistically significant difference from the saline normal        control group. Peptides CMS008 and CMS012, at suitable        concentration, were also found to be able to decrease the        cytotoxic activity of NK cells, having statistically significant        difference from the saline normal control group.    -   3. Peptides CMS001, CMS003, CMS007, CMS009, CMS010, CMS011,        CMS012, CMS015, CMS020, CMS022, and CMS034 were found to be able        to enhance the secretion of interleukin-2 (IL-2) by T        lymphocytes, having statistically significant difference from        the saline normal control group.    -   4. Peptides CMS001 , CMS003, CMS009, CMS010, CMS011, CMS012,        CMS013, CMS016, CMS021, CMS022, and CMS028 were found to be able        to enhance the secretion of IFN by T lymphocytes, having        statistically significant difference form the saline normal        control group.    -   5. Peptides CMS001, CMS002, CMS003, CMS007, CMS008, CMS009,        CMS010, CMS011, CMS012, CMS013, CMS014, CMS015, CMS016, CMS018,        CMS019, CMS020, CMS021, CMS022, CMS023, CMS024, CMS026, CMS027,        CMS028, CMS029, CMS030, CMS032, CMS033, CMS034, CMS035, and        CMS036 were found to be able to enhance the synthesis of        anti-SRBC antibody upon the antigenic challenge, having        statistically significant difference from the saline normal        control group. Peptides CMS002, CMS003, CMS009, CMS010, CMS011,        CMS013, CMS014, CMS015, CMS018, CMS019, CMS020, CMS026, CMS028,        CMS029, CMS030, CMS034, and CMS036, at suitable concentration,        were also found to be able to inhibit the synthesis of anti-SRBC        antibody upon the antigenic challenge, having statistically        significant difference from the saline normal control group.    -   6. Peptides CMS003, CMS008, CMS009, CMS010, CMS011, CMS013,        CMS016, CMS018, CMS019, CMS020, CMS022, CMS024, CMS027, CMS030,        CMS035, CMS036 were found to be able to enhance the phagocytotic        activity of mononuclear phagocyte, having statistic significant        difference from the saline normal control group.    -   7. Peptides CMS001, CMS002, CMS008, CMS010, CMS012, CMS013,        CMS014, CMS015, CMS016, CMS018, CMS019, CMS020, CMS021, CMS022,        CMS023, CMS024, CMS026, CMS027, CMS028, CMS029, CMS030, CMS032,        CMS033, CMS034, CMS035, and CMS036 were found to be able to        increase the weight of the thymus gland, having statistically        significant difference from the saline normal control group.    -   8. Peptides CMS019, CMS020, and CMS030 were found to be able to        increase the weight of the spleen, having statistically        significant difference from the saline normal control group.        Peptides CMS001, CMS003, CMS007, CMS008, CMS009, CMS010, CMS011,        CMS013, CMS014, CMS015, CMS021, CMS023, CMS024, CMS027, CMS029,        and CMS036, at suitable concentration, were also found to be        able to decrease the weight of the spleen, having statistically        significant difference from the saline normal control group.

The materials and methods used to analyze the effect of the peptides onmouse are described below.

Materials

1. Experimental Animal

-   BALB/c Mice, 18-22g weight, 50% female and 50% male, provided by    Experimental Animal Center, National Institute of Medical Science,    PR China.    2. Administration-   recombinant mouse IFN-γ (rmIFN-γ) group: 3×10⁵ IU/kg/day-   recombinant human IL (rhIL)-2 group: 3×10⁵ IU/kg/day-   Saline group: 0.5 ml/each/day-   peptide dose I group: 500 μg/kg/day-   peptide dose II group: 50 μg/kg/day-   peptide dose III group: 5 μg/kg/day-   peptide dose IV group: 0.5 μg/kg/day-   The above substances were all dissolved in 0.5 ml saline and    injected intraperitoneal (i.p.) for 15 continuous days, once per    day.    3. Main Reagents-   The peptides were custom manufactured by American Peptide Company,    Inc., USA-   Fetal bovine serum, and RPMI-1640 cell culture medium, Gibco, USA-   MTT, and ConA, Sigma, USA-   rmIFN-γ, Beijing Biotech Inc., China-   rhIL-2, Shanghai Huaxin Biotech Inc., China-   Lymphocyte separation solution, Research Institute of Hematologic    Disease, National Institute of Medical Science, PR China-   Vesicular Stomatitis Virus (VSV), IFN-γ and IL-2 standard sample,    National Institute For The Control Of Pharmaceutical And Biological    Products, PR China-   HT-2 cell and L929 cell, gift from Prof WF Chen of Beijing    University Department of Immunology, PR China

I. Method

1. The Effect of Peptides on Cellular Immunity

1.1 Preparation of Spleen Cell Suspension^([1,2])

The BALB/c mice were randomly divided into the peptide, IFN, IL-2, andsaline groups. Ten mice per group. The day after the last test substanceadministration, the mice were sacrificed by cervical dislocation. Thespleen was isolated aseptically and manually dispersed in cold D-Hank'ssolution using an injection needle. The dispersed cell suspension wasfurther sieved through a 100 gauge 150 μm diameter stainless steelsieve. After centrifugation at 200 g for 10 minutes, the supernatant wasdiscarded. The cell pellet was re-suspended in 10 volume of Tris-NH₄Clbuffer and then kept standing still for 10 minutes at room temperature.The suspended cells were collected by centrifugation at 150 g for 10minutes. The cells were washed 2-4 times with cold D-Hank's solution byre-suspending and collecting by centrifugation with condition asdescribed above. The washed cells were then diluted to the desired celldensities by RPMI-1640 culture medium, containing 10% fetal bovineserum.

1.2 The Effect of Peptides on T Lymphocyte Transformation^([1,2])

Spleen cells of density 1×10⁶/ml were placed onto a 96 wells cellculture plate, 100 μl/well, three parallel wells each of the assaysample and control sample per mouse. To the assay wells, 100 μl/wellConA of 100 μg/ml in RPMI-1640 was added, and 100 μl/well plainRPMI-1640 was used for the controls. The cells were incubated for 66 hrsat 37° C., 5% CO₂. The cells were then pelleted by centrifugation at 150g for 10 minutes. The supernatant was collected and stored at −20° C.for cytokines IL-2 and IFN determination.

50 μl/well MTT of 1 mg/ml in RPMI-1640 was added to the cell pellet andthe cells re-suspended by shaking for 2 minutes. The incubation wascontinued for 4 hours. The supernatant was discarded aftercentrifugation at 150 g for 10 minutes. 120 μl 40 mM HCl-2-propanol wasadded to the cell pellet and shaken for 3 minutes to obtain OD₅₇₀ nm ofeach well referenced at 630 nm. An ELISA reader was used

Calculation:

Each mouse formed three assay wells and three control wells. TheStimulation Index (SI) of each mouse was obtained by first deriving theaverage OD of the three parallel wells, then dividing the value of theassay wells by the control wells.

1.3 The Effect of Peptides on NK Cell Activity^([3, 4])

Mice spleen cells were prepared to 4×10⁶/ml as described in section 1.1above. Target cells YAC-1 were brought to log phase and adjusted to1×10⁵/ml. Using a 96 wells cell culture plate, 100 μl mouse spleen cellsand 100 μl culture medium were added to the control well containing onlythe spleen cells; 100 μl target cells and 100 μl culture medium wereadded to the control well containing only target cells; 100 μl mousespleen cells and 100 μl target cells were added to the NK activity assaywell. Three parallel sets of the above were prepared per mouse.

Samples were centrifuged at 150 g for 10 minutes to collect the cells.The supernatant was discarded and 50 μl/well MTT of 1 mg/ml was added.The reaction-mixture was then shaken for 2 minutes, and incubated at 37°C., 5% CO₂ for 4 hours. The supernatant was discarded aftercentrifugation at 150 g for 10 minutes. 120 μl 40mM HCl-2-propanol wasadded and shaken for 3 minutes. An ELISA reader was used to obtainOD_(570 nm) of each well referenced at 630 nm.

Calculation:

Each mouse has 9 wells: three spleen cells only control, three targetcells only control, and three assay wells with both spleen and targetcells. The NK cell activity index of each mouse was obtained by firstderiving the average OD of the three parallel wells of each combination,then applying this average OD to the following formula:NK cell activity index=[1−(average OD of spleen and target cellwell−average OD of spleen cell only well)÷(average OD of target cellonly well)]×100%1.4 The Effect of Peptides on the Activity T Lymphocyte in SecretingIL-2^([5])

HT-2 cells at log phase were collected by centrifugation at 150 g for 10minutes, and washed three times with cold Hank's solution byre-suspension and centrifugation. The collected HT-2 cells werere-suspended in RPMI-1640 and incubated at 37° C., 5% CO₂ for 30minutes. The cells were further washed twice with RPMI-1640 byre-suspension and centrifugation, and re-suspended to finalconcentration of 2×10⁵/ml with RPMI-1640.

The supernatant obtained in section 1.2 were diluted to the followingpercentage with RPMI-1640: 100%, 50%, 25%, 12.5%, 6.25%, and 3.125%.

rIL-2 was diluted to the following concentration with RPMI-1640: 500IU/ml, 250 IU/ml, 125 IU/ml, 62.5 IU/ml, 31.25 IU/ml, and 15.5 IU/ml.

A 96 well cell culture plate was set up with three parallel wells percombination:

-   -   Negative control: 100 μl RPMI-1640+100 μl HT-2 cell suspension    -   rIL-2 standard: 100 μl rIL-2 solution+100 μl HT-2 cell        suspension    -   Assay well: 100 μl diluted supernatant+100 μl HT-2 cell        suspension

The plate was incubated at 37° C., 5% CO₂ for 68 hours, then centrifugedat 150 g for 15 minutes and the supernatant removed. 100 μl 0.5 mg/mlMTT in RPMI-1640 without phenolsulfonphthalein was added into each well.After shaking for 3-4 minutes to re-suspend the cells, continue toincubate for another 4 hours. Samples were then centrifuged at 150 g for15 minutes and the supernatant was removed. To each well, 120 μl 40mMHCl-2-propanol was added, mixed for 3-4 minutes and OD analyzed at 570nm, referenced at 630 nm with an ELISA plate reader.

Calculation:

The average OD of the three parallel wells of each dilution was takenand plotted against concentration on a semi-log paper, concentration onthe X-axis. The concentration at 50% OD saturation was obtained for boththe testing supernatant and rIL-2.

Sample IL-2 activity=(sample dilution at 50% maximum action÷rIL-2standard dilution at 50% maximum action)×activity of rIL-2 standard at50% maximum action (IU/ml)

1.5 The Effect of Peptides on the Activity of T Lymphocyte in SecretingInterferon (IFN)^([6])

The supernatant from the section 1.2 was diluted with RPMI-1640 culturemedium to the following percentages: 100%, 50%, 25%, 12.5%, 6.25%, and3.125%.

The recombinant interferon (rIFN) standard was diluted with RPMI-1640 tothe following concentrations: 500 IU/ml, 250 IU/ml, 125 IU/ml, 62.5IU/ml, 31.25 IU/ml, and 15.5 IU/ml.

Target cells L929 at log phase were adjusted to 2×10⁵/ml with RPMI-1640,with treatment same as the HT-2 cell described in section 1.4. Stock VSVwas also adjusted to 100 TCID₅₀ with RPMI-1640.

The following on a 96 well culture plate was set up, three parallelwells per combination:

-   -   Negative control well: 150 μl RPMI-1640+100 μl L929    -   Positive control well: 100 μl RPMI-1640+100 μl L929+50 μl VSV    -   rIFN activity well: 100 μl rIFN standard+100 μl L929+50 μl VSV    -   assay well: 100 μl diluted supernatant+100 μl L929+50 μl VSV

Samples were incubated at 37° C., 5% CO₂ for 24 hours. The positivecontrol wells were observed periodically under inverted microscope toconfirm cell lysis, then collected, washed, and OD of all wells was readsame as described in section 1.4.

Calculation:

Concentrations at 50% maximum action were obtained same way as section1.4. Calculate IFN activity of sample as following:

Sample IFN activity=(sample dilution at 50% maximum action÷rIFN standarddilution at 50% maximum action)×standard rIFN activity at that 50%maximum action (IU/ml)

2. The Effect of Peptides on Antibody Formation^([7])

Sheep red blood cells (SRBC) were prepared by collecting blood fromcervical vein and put into a sterile flask with glass beads. The flaskwas shaken for 3 minutes and the blood then mixed with Alsever solution(glucose 2.05 g, NaCl 0.4 g, Na lemonade 0.8 g, adjust to 100 ml withdistilled water) and stored at 4° C. Immediately before use, sampleswere centrifuged at 130 g, 5 minutes to collect the SRBC. The cells werewashed two times by re-suspension and centrifugation in normal saline.Then the cell pellet was collected by centrifugation at 180 g for 10minutes and re-suspended in saline to make the final working SRBCsuspension, 2% (v/v).

Complement was prepared by adding 10 volumes of fresh Cavy serum intoone volume centrifuge packed SRBC, and then gently shaking for 30minutes at 4° C. The SRBC was removed by centrifugation at 200 g for 10minutes. 10 volumes of normal saline were added to obtain the workingcomplement solution.

The BALB/c mice were randomly divided into the peptide group, IFN group,IL-2 group, and saline group, 10 mice per group. The test substanceswere administered as described in section 1.1, plus intraperitonealinjection 0.2 ml SRBC per mouse on day 12. On the day after the lasttest substance administration (day 16), blood was collected from the eyecanthus and left at room temperature for one hour for serum exudation.After centrifugation at 200 g for 10 minutes, the serum was diluted by500 times with normal saline.

To 1 ml diluted mouse serum of each mouse, 0.5 ml SRBC suspension wasadded. Ice cold. Then 1 ml working complement solution was added andincubated at 37° C. water bath for 10 minutes. Reactions were terminatedby ice cold. Samples were then centrifuged at 200 g for 10 minutes toobtain the supernatant.

To 1 ml of this supernatant, 3 ml Drabkin solution was added and left atroom temperature for 10 minutes. OD_(540 nm) was obtained.

Calculation:

Reference OD_(540 nm) was obtained by mixing 0.25 ml SRBC suspensionwith Drabkin solution to 4 ml and placed at room temperature for 10minutes before OD_(540 nm) was taken.

Sample serum index=(OD_(540 nm) of test sample÷referenceOD_(540 nm))×500 3. The effect of peptides on the phagocytosis functionof mononuclear phagocyte and the weight of immune organ^([8,9]).

On the next day after the last test substance administration (day 16),the mice were injected with 0.1 ml/kg body weight India ink (5 timesdilution with normal saline) from the tail vein.

One minute and five minutes after Indian ink injection, 20 μl blood wasobtained from the eye canthus with a heparinized tubing. The blood wasmixed with 2 ml 0.1% w/v Na₂CO₃ and then OD_(680 nm) obtained. Theoutline clear index K was calculated by the following formula:K=(1 g A ₁−1 g A ₂)÷(t2−t1)

-   -   Key:    -   A1: OD680 nm at first minute    -   A2: OD680 nm at fifth minute    -   t2: 5 minutes    -   t2: 1 minute

One day after the last test substance administration (day 16), theliver, spleen, and thymus gland were separated and blotted dry withfilter paper and weighed. The phagocytosis index cc was calculated asbelow:α=(³ √{square root over ( )}K)(W÷W _(LS))

-   -   key:    -   W: body weight    -   W_(LS): weight of liver plus spleen    -   Thymus gland index (%)=(thymus weight/body weight)×100%    -   Spleen index (%)=(spleen weight/body weight)×100%

Results

Due to the large quantity of raw data involved, only the compiled endresult are presented. The groups without statistically significantdifference from the saline negative control are also omitted.

1. The Effect of Peptides on T Lymphocyte Transformation

At 500 μg/kg/day, CMS002, CMS007, CMS008, CMS009, CMS010, CMS012,CMS015, CMS019, CMS021, and CMS029 were found to be able to enhance Tlymphocyte transformation, having statistically significant differencefrom the saline group (P<0.05). Among these peptides, CMS010 and CMS015were found to have statistically significant difference from the IFN-γgroup and IL-2 group (P<0.05) as shown in Table 1 below. TABLE 1 Group NX ± SD (stimulation index) CMS002 8 1.8 ± 0.3* CMS007 9 1.6 ± 0.1*CMS008 9 1.7 ± 0.1* CMS009 10 1.7 ± 0.2* CMS010 9 2.0 ±0.3*^(@){circumflex over ( )} CMS012 9 1.6 ± 0.2* CMS015 9 1.9 ±0.3*^(@){circumflex over ( )} CMS019 9 1.8 ± 0.3* CMS021 10 1.6 ± 0.1*CMS029 9 1.7 ± 0.3* IFN-γ 10 1.6 ± 0.2* IL-2 10 1.7 ± 0.2* Saline 10 1.3± 0.1*comparing to saline group P < 0.05^(@)comparing to IFN-γ group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.05

At 50 μg/kg/day, CMS001, CMS002 and CMS003 were found to be able tostimulate T lymphocyte transformation, having statistically significantdifference from saline group, IFN-γ group, and IL-2 group (P<0.05).CMS014 and CMS036 were found to be able to inhibit T lymphocytetransformation, having statistical significant difference from salinegroup (P<0.05). Data detailed in Table 2 below. TABLE 2 Group N X ± SD(stimulation index) CMS001 10 2.2 ± 0.5*^(@){circumflex over ( )} CMS00210 2.6 ± 0.3*^(@){circumflex over ( )} CMS003 8 2.2 ±0.5*^(@){circumflex over ( )} CMS014 9 1.0 ± 0.1* CMS036 9 1.0 ± 0.1*IFN-γ 9 1.7 ± 0.2* IL-2 10 1.8 ± 0.2* Saline 10 1.3 ± 0.1*comparing to saline group P < 0.05^(@)comparing to IFN-γ group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.05

At 5 μg/kg/day, CMS010, CMS003, CMS007, and CMS034 were found to be ableto stimulate T lymphocyte transformation, having statisticallysignificant difference from the saline group (P<0.05) as shown in Table3. TABLE 3 Group N X ± SD (stimulation index) CMS001 10 1.7 ± 0.2*CMS003 10 1.6 ± 0.2* CMS007 8 1.7 ± 0.1* CMS034 9 1.5 ± 0.2* IFN-γ 101.6 ± 0.2* IL-2 9 1.6 ± 0.1* Saline 10 1.3 ± 0.1*comparing to saline group P < 0.05

At 0.5 μg/kg/day, CMS008, CMS010, and CMS011 were found to be able tostimulate T lymphocyte transformation, having statistically significantdifference from the saline group (P<0.05) as shown in Table 4. TABLE 4Group N X ± SD (stimulation index) CMS008 10 1.7 ± 0.3* CMS010 9 1.7 ±0.3* CMS011 10 1.6 ± 0.4* IFN-γ 10 1.6 ± 0.2* IL-2 10 1.6 ± 0.1* Saline10 1.3 ± 0.1*comparing to saline group P < 0.05

At 500 μg/kg/day, CMS010, CMS013, CMS016, CMS023, CMS024, CMS026,CMS027, CMS028, CMS029, CMS030, CMS032, CMS033, CMS034, CMS035, andCMS036 were found to be able to increase NK cell cytotoxic activity,with statistically significant difference from the saline group(P<0.05). Among these peptides, CMS010, CMS016, and CMS030 were found tohave statistically significant difference from the IFN-γ group and IL-2group (P<0.05) as shown in Table 5. TABLE 5 Group N X ± SD (%) CMS010 991 ± 4*^(@){circumflex over ( )} CMS013 8 84 ± 9* CMS016 9 91 ±7*^(@){circumflex over ( )} CMS023 10 79 ± 12* CMS024 10 89 ± 8* CMS02610 89 ± 7* CMS027 10 88 ± 8* CMS028 10 90 ± 5* CMS029 10 87 ± 4* CMS03010 91 ± 5*^(@){circumflex over ( )} CMS032 10 87 ± 5* CMS033 9 89 ± 8*CMS034 11 85 ± 9* CMS035 8 90 ± 10* CMS036 10 88 ± 7* IFN-γ 10 77 ± 8*IL-2 10 77 ± 8* Saline 8 63 ± 9*comparing to saline group P < 0.05^(@)comparing to IFN-γ group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.05

At 50 μg/kg/day, CMS001, CMS003, CMS015, CMS021, CMS026, and CMS035 werefound to be able to increase NK cell cytotoxic activity, havingstatistically significant difference from the saline group (P<0.05).Among these peptides, CMS021 was found to have statistically significantdifference from the IFN-γ group and IL-2 group (P<0.05). CMS012 wasfound to be able to inhibit NK cell cytotoxic activity, havingstatistically significant difference from the saline group (P<0.05).Data detailed in Table 6 below. TABLE 6 Group N X ± SD (%) CMS001 10 85± 10* CMS003 10 85 ± 6* CMS012 9 40 ± 9* CMS015 8 78 ± 8* CMS021 8 88 ±12*^(@){circumflex over ( )} CMS026 10 76 ± 9* CMS035 10 72 ± 9* IFN-γ10 73 ± 10* IL-2 10 74 ± 8* Saline 10 56 ± 8*comparing to saline group P < 0.05^(@)comparing to IFN-γ group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.05

At 5 μg/kg/day, CMS008, CMS009, CMS010, CMS011, CMS012, CMS020, CMS024,CMS034, and CMS036 were found to be able to increase NK cell cytotoxicactivity, having statistically significant difference from the salinegroup (P<0.05). Among these peptides, CMS008 and CMS009 were found tohave statistically significant difference from the IFN-γ group and IL-2group (P<0.05) as shown in Table 7. TABLE 7 Group N X ± SD (%) CMS008 1092 ± 4*^(@){circumflex over ( )} CMS009 8 92 ± 6*^(@){circumflex over( )} CMS010 10 82 ± 9* CMS011 10 76 ± 10* CMS012 10 85 ± 7* CMS020 9 91± 6* CMS024 9 78 ± 3* CMS034 8 90 ± 5* CMS036 10 75 ± 9* IFN-γ 10 80 ±8* IL-2 10 80 ± 8* Saline 10 60 ± 9*comparing to saline group P < 0.05^(@)comparing to IFN-γ group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.05

At 0.5 μg/kg/day, CMS002, CMS011, CMS012, CMS022, CMS028, and CMS035were found to be able to increase NK cell cytotoxic activity, havingstatistically significant difference from the saline group (P<0.05).CMS008 was found to be able to inhibit NK cell cytotoxic activity,having statistical significant difference from the saline group(P<0.05). Data detailed in Table 8 below. TABLE 8 Group N X ± SD (%)CMS002 8 76 ± 9* CMS008 10 46 ± 12* CMS011 9 79 ± 3* CMS012 9 77 ± 6*CMS022 10 73 ± 11* CMS028 8 79 ± 3* CMS035 10 76 ± 10* IFN-γ 10 72 ± 9*IL-2 10 74 ± 10* Saline 11 58 ± 7*comparing to saline group P < 0.05

3. The Effect of Peptides on the Activity of T Lymphocyte in SecretingIL-2

At 500 μg/kg/day, CMS007, CMS009, CMS010, and CMS015 were found to beable to promote IL-2 secretion from T lymphocyte, having statisticallysignificant difference from the saline group (P<0.05). Among thesepeptides, CMS007 and CMS015 were found to have statisticallysignificantly difference from the IL-2 group (P<0.05). And, CMS009 andCMS010 were also found to have statistically significant difference fromboth the IFN-γ group and IL-2 group (P<0.05) as shown in Table 9. TABLE9 Group N X ± SD (IU) CMS007 9  86 ± 15*{circumflex over ( )} CMS009 10114 ± 13*^(@){circumflex over ( )} CMS010 9 125 ± 17*^(@){circumflexover ( )} CMS015 9  85 ± 17*{circumflex over ( )} IFN-γ 10 100 ± 18*IL-2 10  70 ± 13* Saline 10  39 ± 10*comparing to saline group P < 0.05^(@)comparing to IFN-γ group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.05

At 50 μg/kg/day, CMS001 and CMS003 were found to be able to promoteactivity of T lymphocyte in secreting IL-2, having statisticallysignificant difference from the saline group (P<0.05). Among thesepeptides, CMS003 was found to have statistically significant differencefrom the IL-2 group (P<0.05) as shown in Table 10. TABLE 10 Group N X ±SD (IU) CMS001 10 60 ± 10* CMS003 8 86 ± 9*{circumflex over ( )} IFN-γ 999 ± 16* IL-2 10 72 ± 12* Saline 10 39 ± 10*comparing to saline group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.05

At 5 μg/kg/day, CMS007, CMS012, and CMS020 were found to be able topromote T lymphocyte in secreting IL-2, having statistically significantdifference from the saline group (P<0.05) as shown in Table 11. TABLE 11Group N X ± SD (IU) CMS007 8 64 ± 12* CMS012 9 65 ± 16* CMS020 8 63 ±11* IFN-γ 10 96 ± 14* IL-2 10 77 ± 13* Saline 10 37 ± 9 *comparing to saline group P < 0.05

At 0.5 μg/kg/day, CMS010, CMS011, CMS012, CMS022, and CMS034 were foundto be able to promote T lymphocyte in secreting IL-2, havingstatistically significant difference from the saline group (P<0.05).Among these peptides, CMS034 was found to have statisticallysignificantly difference from the IL-2 group (P<0.05). And, CMS011 andCMS022 were also found to have statistically significant difference fromboth the IFN-γ group and IL-2 group (P<0.05) as shown in Table 12. TABLE12 Group N X ± SD (IU) CMS010 9  66 ± 11* CMS011 10 101 ±19*^(@){circumflex over ( )} CMS012 8  59 ± 13* CMS022 9 109 ±14*^(@){circumflex over ( )} CMS034 10  85 ± 10*{circumflex over ( )}IFN-γ 10  87 ± 15* IL-2 10  73 ± 13* Saline 10  38 ± 13*comparing to saline group P < 0.05^(@)comparing to IFN-γ group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.05

4. The Effect of Peptides on the Activity of T Lymphocyte in SecretingIFN

At 500 μg/kg/day, CMS010, CMS013, and CMS016 were found to be able topromote T lymphocyte in secreting interferon (IFN), having statisticallysignificant difference from the saline group, IFN-γ group, and IL-2group (P<0.05) as shown in Table 13. TABLE 13 Group N X ± SD (IU) CMS0109 167 ± 13*^(@){circumflex over ( )} CMS013 9 154 ± 15*^(@){circumflexover ( )} CMS016 6 162 ± 19*^(@){circumflex over ( )} IFN-γ 10 139 ± 16*IL-2 10 120 ± 13* Saline 10  65 ± 11*comparing to saline group P < 0.05^(@)comparing to IFN-γ group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.05

At 50 μg/kg/day, CMS001, CMS003, and CMS021 were found to be able topromote T lymphocyte in secreting IFN having statistically significantdifference from the saline group (P<0.05). Among these peptides, CMS021was found to have statistically significant difference from the IFN-γgroup and IL-2 group (P<0.05) as shown in Table 14. TABLE 14 Group N X ±SD (IU) CMS001 10 110 ± 15* CMS003 8 106 ± 16* CMS021 8 143 ±17*^(@){circumflex over ( )} IFN-γ 9 125 ± 18* IL-2 10 113 ± 17* Saline10  61 ± 11*comparing to saline group P < 0.05^(@)comparing to IFN-γ group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.05

At 5 μg/kg/day, CMS009 and CMS012 were found to be able to promote Tlymphocyte in secreting IFN having statistically significant differencefrom the saline group (P<0.05). Among these peptides, CMS009 was foundto have statistically significant difference from the IL-2 group(P<0.05) as shown in Table 15. TABLE 15 Group N X ± SD (IU) CMS009 10121 ± 15*{circumflex over ( )} CMS012 9  86 ± 9* IL-2 9 105 ± 14* Saline10  66 ± 10*comparing to saline group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.05

At 0.5 μg/kg/day, CMS010, CMS011, CMS022, and CMS028 were found to beable to promote T lymphocyte in secreting IFN, having statisticallysignificant difference from the saline group (P<0.05). Among thesepeptides, CMS010 and CMS022 were found to have statistically significantdifference from the IFN-γ group and IL-2 group (P<0.05) as shown inTable 16. TABLE 16 Group N X ± SD (IU) CMS010 9 142 ± 18*^(@){circumflexover ( )} CMS011 10  89 ± 18* CMS022 9 145 ± 13*^(@){circumflex over( )} CMS028 10  96 ± 13* IFN-γ 10 124 ± 16* IL-2 10 107 ± 13* Saline 10 64 ± 13*comparing to saline group P < 0.05^(@)comparing to IFN-γ group P < 0.05{circumflex over ( )}comparing to IL-2 groupP < 0.05

5. The Effect of Peptides on Antibody Formation

At 500 μg/kg/day, CMS002, CMS003, CMS007, CMS008, CMS009, CMS010,CMS011, CMS012, CMS013, CMS014, CMS015, CMS016, CMS018, CMS019, CMS020,CMS022, CMS023, CMS024, CMS029, CMS033, and CMS035 were found to be ableto promote anti-SRBC antibody formation, having statisticallysignificant difference from the saline group (P<0.05). Among thesepeptides, CMS002, CMS003, CMS007, CMS008, CMS013, CMS019, CMS024, andCMS035 were found to have statistically significant difference from theIFN-γ group (P<0.05). And, CMS009, CMS010, CMS011, CMS012, CMS014,CMS015, CMS016, CMS020, CMS023, CMS029, and CMS033 were also found tohave statistically significant difference from both the IFN-γ group andIL-2 group (P<0.05) as shown in Table 17. TABLE 17 Group N X ± SD (Unit)CMS002 10  87 ± 18*^(@) CMS003 10  96 ± 18*^(@) CMS007 10  69 ± 17*^(@)CMS008 10  82 ± 15*^(@) CMS009 10 113 ± 22*^(@){circumflex over ( )}CMS010 10 112 ± 30*^(@){circumflex over ( )} CMS011 8 188 ±16*^(@){circumflex over ( )} CMS012 8 141 ± 21*^(@){circumflex over ( )}CMS013 10  80 ± 16*^(@) CMS014 10 130 ± 24*^(@){circumflex over ( )}CMS015 10 136 ± 22*^(@){circumflex over ( )} CMS016 8 143 ±38*^(@){circumflex over ( )} CMS018 10  66 ± 16* CMS019 10  91 ± 26*^(@)CMS020 6 155 ± 35*^(@){circumflex over ( )} CMS022 8  68 ± 31* CMS023 9110 ± 45*^(@){circumflex over ( )} CMS024 8  75 ± 29*^(@) CMS029 8 115 ±22*^(@){circumflex over ( )} CMS033 10 143 ± 27*^(@){circumflex over( )} CMS035 10  88 ± 16*^(@) IFN-γ 9  37 ± 10 IL-2 10  71 ± 11* Saline10  32 ± 7*comparing to saline group P < 0.05^(@)comparing to IFN-γ group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.05

At 50 μg/kg/day, CMS003, CMS011, CMS012, CMS013, CMS015, CMS021, CMS022,CMS023, CMS026, CMS027, CMS029, CMS030, CMS032, CMS033, CMS034, CMS035,and CMS036 were found to be able to promote anti-SRBC antibodyformation, having statistically significant difference from the salinegroup (P<0.05). Among these peptides, CMS011, CMS013, and CMS015 werefound to have statistically significant difference from the IFN-γ group(P<0.05). And, CMS021, CMS022, CMS023, CMS026, CMS027, CMS029, CMS030,CMS032, CMS033, CMS034, CMS035, and CMS036 were also found to havestatistically significant difference from both the IFN-γ group and IL-2group (P<0.05). CMS009 was found to be able to inhibit anti-SRBCantibody formation, having statistical significant difference from thesaline group (P<0.05). Data detailed in Table 18 below. TABLE 18 Group NX ± SD (Unit) CMS003 10  52 ± 11* CMS009 8  13 ± 5* CMS011 9  67 ±9*^(@) CMS012 8  50 ± 14* CMS013 8  70 ± 9*^(@) CMS015 10  54 ± 9*^(@)CMS021 9  94 ± 20*^(@){circumflex over ( )} CMS022 9 110 ±16*^(@){circumflex over ( )} CMS023 8  84 ± 11*^(@){circumflex over ( )}CMS026 9  98 ± 9*^(@){circumflex over ( )} CMS027 9  93 ±11*^(@){circumflex over ( )} CMS029 10 143 ± 13*^(@){circumflex over( )} CMS030 10 141 ± 33*^(@){circumflex over ( )} CMS032 9 131 ±24*^(@){circumflex over ( )} CMS033 8 112 ± 15*^(@){circumflex over ( )}CMS034 10 136 ± 11*^(@){circumflex over ( )} CMS035 8  97 ±10*^(@){circumflex over ( )} CMS036 10 118 ± 11*^(@){circumflex over( )} IFN-γ 9  37 ± 10 IL-2 10  71 ± 11* Saline 10  32 ± 7*comparing to saline group P < 0.05^(@)comparing to IFN-γ group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.05

At 5 μg/kg/day, CMS001, CMS003, CMS007, CMS008, CMS009, CMS011, CMS012,CMS013, CMS015, CMS016, CMS019, CMS020, CMS021, CMS023, CMS024, CMS026,CMS027, CMS028, CMS029, CMS030, CMS032, CMS033, CMS034, CMS035, andCMS036 were found to be able to promote anti-SRBC antibody formation,having statistically significant difference from the saline group(P<0.05). Among these peptides, CMS003, CMS008, CMS009, CMS012, CMS013,CMS015, CMS016, CMS020, and CMS021 were found to have statisticallysignificant difference from the IFN-γ group (P<0.05). And, CMS001,CMS007, CMS011, CMS019, CMS023, CMS024, CMS026, CMS027, CMS028, CMS029,CMS030, CMS032, CMS033, CMS034, CMS035, and CMS036 were also found tohave statistically significant difference from both the IFN-y group andIL-2 group (P<0.05) as shown in Table 19. TABLE 19 Group N X ± SD(Unit)CMS001 9 110 ± 24*^(@) CMS003 9  91 ± 24*^(@) CMS007 9 122 ±12*^(@){circumflex over ( )} CMS008 9  97 ± 26*^(@) CMS009 8  79 ±18*^(@) CMS011 10 115 ± 27*^(@){circumflex over ( )} CMS012 10  81 ±22*^(@) CMS013 10  93 ± 28*^(@) CMS015 8  94 ± 37*^(@) CMS016 9  93 ±32*^(@) CMS019 10 118 ± 20*^(@){circumflex over ( )} CMS020 10  89 ±24*^(@) CMS021 9  82 ± 30*^(@) CMS023 10 166 ± 27*^(@){circumflex over( )} CMS024 7 171 ± 39*^(@){circumflex over ( )} CMS026 9 191 ±17*^(@){circumflex over ( )} CMS027 9 117 ± 45*^(@){circumflex over ( )}CMS028 10 121 ± 48*^(@){circumflex over ( )} CMS029 9 147 ±23*^(@){circumflex over ( )} CMS030 9 158 ± 37*^(@){circumflex over ( )}CMS032 9 157 ± 37*^(@){circumflex over ( )} CMS033 7 128 ±39*^(@){circumflex over ( )} CMS034 8 172 ± 37*^(@){circumflex over ( )}CMS035 9 176 ± 39*^(@){circumflex over ( )} CMS036 8 179 ±34*^(@){circumflex over ( )} IFN-γ 9  37 ± 10 IL-2 10  71 ± 11* Saline10  32 ± 7*comparing to saline group P < 0.05^(@)comparing to IFN-γ group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.05

At 0.5 μg/kg/day, CMS021, CMS023, CMS024, CMS027, and CMS033 were foundto be able to promote anti-SRBC antibody formation, having statisticallysignificant difference from the saline group (P<0.05). Among thesepeptides, CMS021 and CMS033 were found to have statistically significantdifference from the IFN-γ group (P<0.05). And, CMS023, CMS024, andCMS027 were also found to have statistically significant difference fromboth the IFN-γ group and IL-2 group (P<0.05). Also, CMS002, CMS003,CMS009, CMS010, CMS011, CMS013, CMS014, CMS015, CMS018, CMS019, CMS020,CMS026, CMS028, CMS029, CMS030, CMS034, and CMS036 were found to be ableto inhibit anti-SRBC antibody formation, having statisticallysignificant difference from the saline group (P<0.05). Data detailed inTable 20 below. TABLE 20 Group N X ± SD (Unit) CMS002 9  4 ± 1* CMS003 9 2 ± 1* CMS009 9  2 ± 1* CMS010 10  10 ± 3* CMS011 10  5 ± 3* CMS013 10 7 ± 1* CMS014 10  15 ± 6* CMS015 9  13 ± 4* CMS018 9  3 ± 1* CMS019 9 12 ± 3* CMS020 9  10 ± 3* CMS021 9  57 ± 9*^(@) CMS023 10 108 ±21*^(@){circumflex over ( )} CMS024 10  98 ± 6*^(@){circumflex over ( )}CMS026 10  19 ± 6* CMS027 10  99 ± 14*^(@){circumflex over ( )} CMS02810  18 ± 5* CMS029 9  18 ± 7* CMS030 9  19 ± 7* CMS033 9  78 ± 12*^(@)CMS034 10  20 ± 2* CMS036 9  20 ± 6* IFN-γ 9  37 ± 10 IL-2 10  71 ± 11*Saline 10  32 ± 7*comparing to saline group P < 0.05^(@)comparing to IFN-γ group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.056. The Effect of Peptides on the Phagocytotic Activity of MononuclearPhagocyte

At 500 μg/kg/day, CMS003, CMS008, CMS020, CMS022, and CMS024 were foundto be able to enhance the phagocytotic activity of mononuclearphagocyte, having statistically significant difference from the salinegroup (P<0.05). Among these peptides, CMS022 was found to havestatistically significant difference from the IFN-γ group and IL-2 group(P<0.05) as shown in Table 21. TABLE 21 Group N X ± SD (phagocytoticindex) CMS003 10 6.6 ± 0.7* CMS008 10 6.5 ± 1.2* CMS020 10 6.4 ± 0.6*CMS022 10 7.4 ± 0.6*^(@){circumflex over ( )} CMS024 10 6.4 ± 1.0* IFN-γ10 6.4 ± 0.9* IL-2 9 5.7 ± 0.8 Saline 10 5.1 ± 0.6*comparing to saline group P < 0.05^(@)comparing to IFN-γ group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.05

At 50 μg/kg/day, CMS019, CMS024, and CMS030 were found to be able toenhance the phagocytotic activity of mononuclear phagocyte, havingstatistically significant difference from the saline group (P<0.05).Among these peptides, CMS019 was found to have statistically significantdifference from the IL-2 group (P<0.05) as shown in Table 22. TABLE 22Group N X ± SD (phagocytotic index) CMS019 9 6.7 ± 0.9*{circumflex over( )} CMS024 8 6.6 ± 0.7* CMS030 10 6.3 ± 0.5* IFN-γ 10 6.4 ± 0.9* IL-2 95.7 ± 0.8 Saline 10 5.1 ± 0.6*comparing to saline group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.05

At 5 μg/kg/day, CMS003, CMS008, CMS009, CMS010, CMS011, CMS013, CMS016,CMS018, CMS019, and CMS035 were found to be able to enhance thephagocytotic activity of mononuclear phagocyte, having statisticallysignificant difference from the saline group (P<0.05). Among thesepeptides, CMS003, CMS009, CMS010, CMS016, CMS019, and CMS035 were foundto have statistically significant difference from the IL-2 group(P<0.05) as shown in Table 23. TABLE 23 Group N X ± SD (phagocytoticindex) CMS003 9 6.9 ± 0.9*{circumflex over ( )} CMS008 9 6.4 ± 0.5*CMS009 9 6.9 ± 0.9*{circumflex over ( )} CMS010 10 7.1 ± 0.7*{circumflexover ( )} CMS011 10 6.4 ± 1.1* CMS013 10 6.7 ± 0.2* CMS016 9 6.9 ±0.8*{circumflex over ( )} CMS018 8 6.7 ± 1.2* CMS019 8 6.8 ±0.6*{circumflex over ( )} CMS035 9 6.9 ± 0.9*{circumflex over ( )} IFN-γ10 6.4 ± 0.9* IL-2 9 5.7 ± 0.8 Saline 10 5.1 ± 0.6*comparing to saline group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.05

At 0.5 μg/kg/day, CMS024, CMS027, and CMS036 were found to be able toenhance the phagocytotic activity of mononuclear phagocyte, havingstatistically significant difference from the saline group (P<0.05).Among these peptides, CMS024 was found to have statistically significantdifference from the IL[-2 group (P<0.05) as shown in Table 24. TABLE 24Group N X ± SD (phagocytotic index) CMS024 10 6.7 ± 0.5*{circumflex over( )} CMS027 10 6.4 ± 0.6* CMS036 9 6.2 ± 0.3* IFN-γ 10 6.4 ± 0.9* IL-2 95.7 ± 0.8 Saline 10 5.1 ± 0.6*comparing to saline group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.057. The Effect of Peptides on the Weight of Immune Organ

At 500 μg/kg/day, CMS008, CMS010, CMS016, CMS019, CMS020, CMS022,CMS026, CMS027, CMS028, CMS029, CMS030, CMS032, CMS033, CMS034, CMS035,and CMS036 were found to be able to increase the weight of thymus gland,having statistically significant difference from the saline group(P<0.05). Among these peptides, CMS027 and CMS034 were found to havestatistically significant difference from the IL-2 group (P<0.05). And,CMS008, CMS022, CMS029, CMS030, CMS032, CMS033, and CMS035 were alsofound to have statistically significant difference from both the IFN-γgroup and IL-2 group (P<0.05) as shown in Table 25. TABLE 25 Group N X ±SD (%) CMS008 8 0.21 ± 0.03*^(@){circumflex over ( )} CMS010 10 0.19 ±0.04* CMS016 9 0.18 ± 0.05* CMS019 10 0.19 ± 0.02* CMS020 10 0.19 ±0.04* CMS022 9 0.26 ± 0.05*^(@){circumflex over ( )} CMS026 10 0.20 ±0.03*{circumflex over ( )} CMS027 8 0.20 ± 0.03*{circumflex over ( )}CMS028 10 0.19 ± 0.02* CMS029 10 0.22 ± 0.04*^(@){circumflex over ( )}CMS030 8 0.30 ± 0.03*^(@){circumflex over ( )} CMS032 8 0.25 ±0.03*^(@){circumflex over ( )} CMS033 9 0.25 ± 0.04*^(@){circumflex over( )} CMS034 9 0.20 ± 0.05*{circumflex over ( )} CMS035 10 0.21 ±0.03*^(@){circumflex over ( )} CMS036 9 0.18 ± 0.02* IFN-γ 10 0.15 ±0.04 IL-2 9 0.14 ± 0.03 Saline 9 0.12 ± 0.02*comparing to saline group P < 0.05^(@)comparing to IFN-γ group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.05

At 500 μg/kg/day, CMS019 was found to be able to increase the weight ofspleen, with statistically significant difference from the salinecontrol groups (P<0.05) as shown in Table 26. CMS001, CMS003, CMS007,CMS009, CMS011, CMS013, CMS014, CMS015, CMS021, CMS023, CMS024, CMS027,and CMS036 were found to be able to decrease the weight of spleen, withstatistically significant difference from the saline control group(P<0.05). Data detailed in Table 26 below. TABLE 26 Group N X ± SD (%)CMS001 10 0.43 ± 0.07* CMS003 8 0.40 ± 0.04* CMS007 9 0.32 ± 0.05*CMS009 9 0.41 ± 0.03* CMS011 9 0.41 ± 0.04* CMS013 10 0.44 ± 0.07*CMS014 10 0.40 ± 0.03* CMS015 9 0.36 ± 0.07* CMS019 9 0.63 ± 0.08*CMS021 9 0.36 ± 0.04* CMS023 9 0.36 ± 0.06* CMS024 9 0.34 ± 0.05* CMS02710 0.37 ± 0.03* CMS036 10 0.40 ± 0.03* Saline 10 0.53 ± 0.05*comparing to saline group P < 0.05

At 50 μg/kg/day, CMS002, CMS008, CMS012, CMS014, CMS016, CMS018, CM019,CMS020, CMS022, CMS023, CMS024, CMS026, CMS027, CMS028, CMS029, CMS030,CMS032, CMS033, CMS034, and CMS036 were found to be able to increase theweight of thymus gland, having statistically significant difference fromthe saline group (P<0.05). Among these peptides, CMS034 was found tohave statistically significant difference from the IL-2 group (P<0.05).And, CMS002, CMS008, CMS012, CMS014, CMS016, CMS018, CMS019, CMS020,CMS022, CMS023, CMS024, CMS026, CMS027, CMS030, CMS032, and CMS036 werealso found to have statistically significant difference from both theIFN-γ group and IL-2 group (P<0.05) as shown in Table 27. TABLE 27 GroupN X ± SD (%) CMS002 10 0.21 ± 0.02*^(@){circumflex over ( )} CMS008 100.20 ± 0.04*^(@){circumflex over ( )} CMS012 10 0.26 ±0.02*^(@){circumflex over ( )} CMS014 10 0.21 ± 0.02*^(@){circumflexover ( )} CMS016 10 0.20 ± 0.03*^(@){circumflex over ( )} CMS018 10 0.23± 0.02*^(@){circumflex over ( )} CMS019 10 0.20 ± 0.03*^(@){circumflexover ( )} CMS020 10 0.27 ± 0.03*^(@){circumflex over ( )} CMS022 10 0.30± 0.03*^(@){circumflex over ( )} CMS023 10 0.20 ± 0.02*^(@){circumflexover ( )} CMS024 10 0.27 ± 0.02*^(@){circumflex over ( )} CMS026 10 0.27± 0.02*^(@){circumflex over ( )} CMS027 8 0.21 ± 0.03*^(@){circumflexover ( )} CMS028 10 0.18 ± 0.04* CMS029 9 0.18 ± 0.05* CMS030 10 0.25 ±0.04*^(@){circumflex over ( )} CMS032 10 0.27 ± 0.03*^(@){circumflexover ( )} CMS033 9 0.18 ± 0.03* CMS034 8 0.19 ± 0.04*{circumflex over( )} CMS036 9 0.22 ± 0.02*^(@){circumflex over ( )} IFN-γ 10 0.15 ± 0.04IL-2 9 0.14 ± 0.04 Saline 9 0.12 ± 0.02*comparing to saline group P < 0.05^(@)comparing to IFN-γ group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.05

At 50 μg/kg/day, CMS008, CMS010, and CMS029 were found to be able todecrease the weight of spleen, with statistically significant differencefrom the saline control group (P<0.05). Data detailed in Table 28 below.TABLE 28 Group N X ± SD (%) CMS008 10 0.39 ± 0.08* CMS010 10 0.38 ±0.05* CMS029 10 0.42 ± 0.04* IFN-γ 10 0.50 ± 0.04 IL-2 9 0.62 ± 0.07Saline 9 0.53 ± 0.05*comparing to saline group P < 0.05

At 5 μg/kg/day, CMS001, CMS002, CMS010, CMS011, CMS012, CMS013, CMS014,CMS015, CMS016, CMS018, CMS019, CMS020, CMS021, CMS022, CMS023, CMS024,CMS026, CMS028, CMS029, CMS030, CMS032, CMS033, CMS034, CMS 035 andCMS036 were found to be able to increase the weight of thymus gland,having statistically significant difference from the saline group(P<0.05). Among these peptides, CMS002, CMS014, CMS024, and CMS030 werefound to have statistically significant difference from the IL-2 group(P<0.05). And, CMS010, CMS012, CMS018, CMS019, CMS020, CMS022, CMS026,CMS028, CMS032, CMS034, and CMS036 were also found to have statisticallysignificant difference from both the IFN-γ group and IL-2 group (P<0.05)as shown in Table 29. TABLE 29 Group N X ± SD (%) CMS001 10 0.22 ± 0.05*CMS002 9 0.24 ± 0.05*{circumflex over ( )} CMS010 9 0.27 ±0.05*^(@){circumflex over ( )} CMS011 10 0.22 ± 0.04* CMS012 10 0.27 ±0.06*^(@){circumflex over ( )} CMS013 10 0.21 ± 0.05* CMS014 10 0.23 ±0.06*{circumflex over ( )} CMS015 9 0.20 ± 0.08* CMS016 10 0.22 ± 0.06*CMS018 10 0.24 ± 0.04*^(@){circumflex over ( )} CMS019 10 0.24 ±0.02*^(@){circumflex over ( )} CMS020 10 0.24 ± 0.07*^(@){circumflexover ( )} CMS021 9 0.20 ± 0.06* CMS022 9 0.25 ± 0.04*^(@){circumflexover ( )} CMS023 10 0.23 ± 0.06* CMS024 9 0.23 ± 0.06*{circumflex over( )} CMS026 10 0.31 ± 0.05*^(@){circumflex over ( )} CMS028 10 0.28 ±0.06*^(@){circumflex over ( )} CMS029 10 0.21 ± 0.03* CMS030 10 0.23 ±0.07*{circumflex over ( )} CMS032 10 0.29 ± 0.04*^(@){circumflex over( )} CMS033 10 0.20 ± 0.02* CMS034 9 0.27 ± 0.06*^(@){circumflex over( )} CMS035 10 0.21 ± 0.04* CMS036 10 0.25 ± 0.04*^(@){circumflex over( )} IFN-γ 10 0.15 ± 0.04 IL-2 9 0.14 ± 0.04 Saline 9 0.12 ± 0.02*comparing to saline group P < 0.05^(@)comparing to IFN-γ group P < 0.05{circumflex over ( )}comparing to IL-2 groupP < 0.05

At 5 μg/kg/day, CMS030 was found to be able to increase the weight ofspleen, having statistically significant difference from the salinegroup (P<0.05). CMS015 was found to be able to decrease the weight ofspleen, having statistically significant difference from the salinegroup (P<0.05). Data detailed in Table 30 below. TABLE 30 Group N X ± SD(%) CMS015 9 0.38 ± 0.15* CMS030 10 0.64 ± 0.09* Saline 10 0.53 ± 0.05*comparing to saline group P < 0.05

At 0.5 μg/kg/day, CMS002, CMS008, CMS010, CMS012, CMS014, CMS018,CMS020, CMS022, CMS026, CMS028, CMS030, and CMS032 were found to be ableto increase the weight of the thymus gland, having statisticallysignificant difference from the saline group (P<0.05). Among thesepeptides, CMS008 and CMS012 were found to have statistically significantdifference from the IL-2 group (P<0.05). And, CMS002, CMS020, and CMS030were also found to have statistically significant difference from boththe IFN-γ group and IL-2 group (P<0.05) as shown in Table 31. TABLE 31Group N X ± SD (%) CMS002 8 0.26 ± 0.06*^(@){circumflex over ( )} CMS00810 0.22 ± 0.07*{circumflex over ( )} CMS010 9 0.21 ± 0.03* CMS012 100.22 ± 0.06*{circumflex over ( )} CMS014 10 0.20 ± 0.04* CMS018 10 0.20± 0.03* CMS020 9 0.23 ± 0.05*^(@){circumflex over ( )} CMS022 10 0.21 ±0.06* CMS026 9 0.21 ± 0.05* CMS028 10 0.20 ± 0.06* CMS030 8 0.24 ±0.05*^(@){circumflex over ( )} CMS032 10 0.21 ± 0.06* IFN-γ 10 0.15 ±0.04 IL-2 9 0.14 ± 0.04 Saline 9 0.12 ± 0.02*comparing to saline group P < 0.05^(@)comparing to IFN-γ group P < 0.05{circumflex over ( )}comparing to IL-2 groupP < 0.05

At 0.5 μg/kg/day, CMS020 was found to be able to increase the weight ofspleen, having statistically significant difference from the salinegroup, IFN-γ group, and IL-2 group (P<0.05). CMS001 was found to be ableto decrease the weight of spleen, having statistically significantdifference from the saline group (P<0.05). Data detailed in Table 32below. TABLE 32 Group N X ± SD (%) CMS001 10 0.40 ± 0.05* CMS020 8 0.68± 0.09*^(@){circumflex over ( )} Saline 10 0.53 ± 0.05 IFN-γ 10 0.50 ±0.04 IL-2 10 0.62 ± 0.07*comparing to saline group P < 0.05^(@)comparing to IFN-γ group P < 0.05{circumflex over ( )}comparing to IL-2 group P < 0.05

In summary, we found that peptides CMS001, CMS002, CMS003, CMS007,CMS008, CMS009, CMS010, CMS011, CMS012, CMS013, CMS014, CMS015, CMS016,CMS018, CMS019, CMS020, CMS021, CMS022, CMS023, CMS024, CMS026, CMS027,CMS028, CMS029, CMS030, CMS032, CMS033, CMS034, CMS035, and CMS036 havein vivo biological activities in the animal model tested.

II. The In Vivo Antiviral Effect of Peptides

In order to find out whether these peptides have possible therapeuticeffect on viral infections, we used the animal model duck hepatitis Binfection in this study to test out the in vivo effects of the abovepeptides on diseased animals.

Chongqing duck hepatitis B model was set up and treated with peptides byintraperitoneal injection (50 μg/kg/day, once per day) for 4 weeks. Theserum level of DHBV DNA was analyzed by serum dot-blot hybridization.Lamivudine and normal saline treatment was used as positive and negativecontrol respectively. The peptide CMS001 was found to be able to reducethe serum level of DHBV DNA at the 4th week of the treatment, havingstatistically significant difference from the saline control group(p<0.05). It is concluded that CMS001 at suitable dosage level can beused as a part, or on its own, for viral hepatitis infection management.

Materials and Methods

1. Peptides were Custom Synthesized by American Peptide Company, Inc.,USA, from L-Amino Acids.

2. Animal Model^([1])

One-day old Chongqing ducks were inoculated with 0.1 ml stock serumpositive of Duck Hepatitis B Virus (DHBV) DNA (5×10⁷ copy/ml) byintraperitoneal injection. One week later, blood samples were collectedfrom the external jugular vein and infection was confirmed by dot-blothybridization with DHBV DNA probe labeled with digoxin^([2]). The duckswere breed to 2 weeks old for entrance into the study.

3. Grouping and Treatment

DHBV infected ducks were randomized into the following groups:

-   -   a) Negative control group (n=9): Normal saline was given 1 ml        per duck by intraperitoneal injection, once per day.    -   b) Lamivudine group (n=8): As positive control group.        Lamivudine^([4]) was given 50 mg/kg/day by oral administration,        once per day.    -   c) Peptide group (n=9): 50 μg/kg/day peptide (adjusted to final        volume of 0.5 to 1 ml with normal saline) was given once per day        by intraperitoneal injection.

Treatment lasted for 4 weeks and observation continued for another oneweek after termination of treatment. 1 ml blood samples were drawn fromthe external jugular vein of the ducks on days 0, 7, 14, 21, 28, and 35when treatment started. Sera of the blood samples were isolatedimmediately^([3]) and stored at −20° C. until analysis.

4. Serum DHBV DNA Level Determinations

DHBV DNA probe was fluorescent labeled according to the labeling kitprotocol from the kit manufacturer (Amersham Pharmacia Biotech Co.). 40μl duck sera were dot-blotted (1 duplicated dot per sample) ontonitro-cellulose membrane and hybridized with fluorescent-labeled DHBVDNA probe for quantitation^([2]). After completion of hybridation, theblots were developed in CDP-Star fluorimetry reagent RPN3690 and scannedwith Vuego Scan (Brisa-620st) scanner. ImageMaster TotalLab v1.11.Inksoftware was used for quantitative analysis of the blots. Statisticalanalysis was carried out according to the pair t-test with SPSSsoftware.

Results TABLE II.1 Serum DHBV DNA titer before and after treatment DHBVDNA level (Mean ± Standard Deviation, 10³ units) Day 0 Day 7 Day 14 Day21 Day 28 Day 35 Normal 26 ± 12 45 ± 31 49 ± 23 102 ± 66  60 ± 38 50 ±43 saline Lamivudine 21 ± 9   6 ± 4*  7 ± 6*  8 ± 7*  8 ± 5* 20 ± 19CMS001 21 ± 18 11 ± 13 20 ± 18 14 ± 14  5 ± 3* 18 ± 16Pair t-test, comparing with day 0 of the same animals:*P < 0.05

The negative (normal saline) and positive (lamivudine) control groupproved successful establishment of the hepatitis animal model. At 50μg/kg/day, the peptide CMS001 was found to be able to decrease the serumDHBV DNA titre after 4 weeks of treatment, having statisticallysignificant difference (p<0.05) from the before treatment value of thesame animals. Upon termination of treatment, the serum DHBV DNA titrerebounced to a value with no statistical difference from that beforetreatment, showing that the effect of peptide CMS001 may be reversibleand/or need longer treatment period for eradication of the virus.

Discussion

Duck hepatitis animal model^([1]) is an established experimental modelfor human hepatitis B pathogenesis studies and for the screening ofhepatitis B therapeutic agents. In this study, CMS001 was found to beable to decrease the serum titre of DHBV DNA after 4 weeks of treatment,indicating that the peptide CMS001 at suitable dosage level and withsuitable application scheme, can be useful on its own or in combinationwith other substances as an agent for human hepatitis B management.

In the present study, administration by intraperitoneal injection wastested, but this does not exclude the possible effectiveness of thepeptide if administered via other alternative routes. The peptides mayalso be administered via intravenous injection, intramuscular injection,subcutaneous injection, and subcutaneous implantation, with or withoutdelivery facilitating device such as liposome, sustain releaseprotection etc. The peptide may also be administered in any form of oraladministration like tablet, capsule, suspension, solution etc, in theusual form without modification or in slow release form, or with orwithout gastro-enteric protection. The peptide may further be applied inany form of topic application like ointment, cream, gel etc with orwithout transdermal facilitating device, or as inhalant of powder,dissolved, or as liposome protected form. The peptide may also beinterpreted into its genetic sequence and cloned into an expressionsystem, on its own or in combination with other peptide sequences, togenerate a resulting peptide molecule to make use of the activity of thepeptide as described in this report, with or without purification of theresulting peptide. TABLE II.2 Peptides Effective for Hepatitis B CMScode SEQ ID No. CMS001 1References1. Chen Yaxi, Guo shuhua, Zhang Dingfeng, et al. Foundation andapplication of Chongqing duck hepatitis B model. Chinese Journal ofHepatology. 1993; 1(2): 89-912. Chen Yaxi, Guo shuhua, Chen Xuehua. Preparation and application ofDHBV DNA probe labeled with digoxin. Journal of Chongqing University ofMedical Sciences. 1994; 19(4): 295-2973. Tang Ni, Huang Ailong, Guo shuhua, et al. Systemic foundation andapplication of serological parameters of humoral immunity to duckhepatitis B virus. Chinese Journal of Hepatology. 2001; 9(1): 13-154. Chen Yaxi, Guo shuhua, Qi Zhenyuan, et al. An experimental study oflamivudine against duck hepatitis B virus in combination withfamciclovir. Chinese Journal of Hepatology. 2001; 9(4): 209-211III. Effect of Peptides on Nephritis

In order to find out whether these peptides have possible therapeuticeffect on nephritis, we used the animal model rat Masugi nephritis inthis study to test out the in vivo effects of the above peptides ondiseased animals^([3,4]).

The objective of this study is to investigate the in vivo therapeuticeffect of peptides on chronic glomerulonephritis. Masugi nephritis ratmodel was constructed by injecting rabbit anti-rat-renal-cortex IgG intoheathy Sprague Dawley rats, and then treated immediately withintraperitoneal injection of peptides at 50 μg/kg/day, once per day for3 weeks. Hydrocortisone was used as positive control. It was found thatproteinurea, serum creatinine level, and splenic index of the ratstreated with CMS014, CMS018, CMS030, and CMS036 were lowered comparedwith that of the control group, with statistical significance (p<0.05).Postmortum microscopic examination of the kidney showed that thetherapeutic effect of these peptides was similar to hydrocortisonetreatment. It is concluded that CMS014, CMS018, CMS030, and CMS036 maybe used as a means for chronic nephritis management.

Materials

Sprague Dawley (SD) rats, male, weighing 120±20 g, were from Center ofExperimental Animal, Guangzhou University of Traditional ChineseMedicine, and also from First Military Medical University. Chinchillarabbits weighing 3 kg were from Center of Experimental Animal, GuangzhouUniversity of Traditional Chinese Medicine.

Peptides, of L-amino acids origin, were custom synthesized by AmericanPeptide Company, Inc, USA, and were diluted to 10 μg/ml in sterilenormal saline. Hydrocortisone was from Yangzhou Pharmaceutical Factory,China.

Serum creatinine level determination kit from Shanghai RongshengBiological Technique Company, PR China.

Methods

1. Preparation of Rabbit Anti-Rat-Renal-Cortex Anti-Serum^([1]):

20 healthy SD rats were anesthetized with intravenous injection of 3%sodium pentobarbital, 40 mg/kg. The aorta abdominalis was exposed andthe kidneys were perfused with normal saline until clean of blood. Therenal cortex was isolated, homogenized in 5 volumes of 0.01M Tris-HClbuffer, pH 8.1, and filtered through a 140-gauge stainless steel mesh.The filtrate was collected and mixed with either GIBCO-RRE complete orincomplete Freunds adjuvant in 1:1 and emulsified to obtain the workingimmunization solution.

10 healthy rabbits were immunized with the immunization solutions, firsttime with complete Freunds adjuvant and subsequently with the incompleteone. The antigen was injected hypodermally through 6 random points, 0.1ml per point, once per 10 days. From 6^(th) week onwards, blood wascollected from the auricular vein and the antibody titer was determinedby the double diffusion method^([2]). On the 8^(th) week after startingof immunization, the rabbits were anesthetized with intravenousinjection of 3% sodium pentobarbital, 20 mg/kg, and blood was collectedfrom the carotid artery. The anti-serum was then exuded and isolated.

Whole blood was collected from 15 healthy SD rats. The red blood cellwas isolated and washed clean thrice with normal saline. The clean redblood cell was mixed with 250 ml rabbit anti-serum and incubated at 4°C. overnight. After incubation, the blood cell was removed bycentrifugation and the supernatent was further inactivated at 56° C. for30 minutes. After centrifugation to remove any precipitate, crude IgGwas isolated and partially purified from the supernatent byprecipitation with (NH₄)₂SO₄ thrice (50%, 33%, and then 33%)^([5]). Thecrude IgG was redissolved in 125 ml double distilled water and dialysedclean of ammonium sulphate. The anti-rat-renal-cortex antibody titer wasdetermined by the double diffusion method^([2]).

2. Induction of Rat Glomerulonephritis^([5]):

0.25 ml priming solution (containing about 4 mg non-specific rabbit IgGwith complete Freunds adjuvant) was injected intraperitoneally intohealthy SD rats to prime the rats five days before induction. With theexception of the normal healthy control group that received normalsaline, all groups were subsequently induced by intraperitonealinjection of 1 ml rabbit anti-rat-renal-cortex IgG prepared as describedin section Method 1.

3. Grouping and Administration:

Male SD rats were randomized into groups of normal healthy control(normal), nephritis placebo treatment control (placebo), nephritispeptide treatment, and the nephritis hydrocortisone treatment control(hydrocortisone), 12 per group. Treatment started on the day ofinduction. Peptides 50 μg/kg/day, hydrocortisone 3.3 mg/kg/day, andnormal saline was used as placebo, all administered intraperitoneallyonce per day and lasted for 3 weeks.

4. Parameters Monitored^([4]):

-   -   (a) urine protein level: each rat was kept individually in one        cage. Adequate drinking water was supplied. Urine was collected        once per week, 24 hours per time. Protein content of the urine        was determined by the Coomassie blue method.    -   (b) serum creatinine level: blood was collected after three        weeks of treatment and serum creatinine level determination. The        creatinine kit was provided by Shanghai Rongsheng Biological        Technique Company.    -   (c) Spleen weight index: spleen weight index was determined by        the following formula:        Spleen weight index=mean spleen weight÷mean body weight    -   (d) Pathological microscopic examination of kidney: 6 heaviest        kidneys were chosen from each group for pathological        examination.        Statistical Analysis

Used t-test for inter-group comparisons, cut off set at p<0.05. For allgroups, the two rats with lowest urinary protein levels were excludedfrom the statistical analysis.

Result

1. The Effect of Peptide Treatment on the Urine Protein Level TABLEIII.1 The effect of peptide treatment on urine protein level (unit: mg)Groups n Week 1 Week 2 Week 3 CMS014 10 5.5 ± 4.0* 6.3 ± 6.8* 2.8 ± 1.9*CMS018 10 7.0 ± 3.7* 5.5 ± 7.9* 3.3 ± 3.4* CMS030 10 5.4 ± 3.4* 9.5 ±16.2 2.4 ± 1.5* CMS036 10 7.9 ± 5.8* 5.0 ± 7.1* 1.3 ± 0.9*hydrocortisone 10 3.1 ± 2.0* 7.5 ± 7.7  7.6 ± 7.1* placebo 10 22.9 ±22   17.2 ± 14.5  20.2 ± 29.0  normal 9 1.7 ± 1.3* 2.3 ± 1.1* 0.4 ± 0.2*Comparing with placebo, *p < 0.05

It was found that peptides CMS014, CMS018, CMS030, and CMS036 at 50μg/kg/day once per day can lower the urine protein level of Masuginephritis rat model, with statistically significant difference from theplacebo treated control group, p<0.05. It was also noted that the growthrate of groups CMS014, CMS030, CMS036, and hydrocortisone have slowergrowth rate than normal. The growth rate of the hydrocortisone groupdecreased to such an extent that after the first week, the treatmentdose has to be halved to avoid severe intolerance.

2. The Effect of Peptide Treatment on the Serum Creatinine Level TABLEIII.2 The influence of peptides on the serum creatinine level Groups nSerum creatinine level (μmol/L) CMS014 10 159 ± 1  CMS018 10 67 ± 1*CMS030 10 93 ± 1* CMS036 10 80 ± 1* Placebo 10 265 ± 212 hydrocortisone10 239 ± 107 normal 9 80 ± 1*Comparing with nephritis rats receiving placebo treatment (placebo), *p< 0.05

The serum creatinine level of Masugi nephritis rats receiving placebotreatment was much higher than that of the normal control group, showingthat the renal function of the nephritis rats was abnormal. It was foundthat the peptides CMS014, CMS018, CMS030, and CMS036 at 50 μg/kg/dayonce per day were able to lower the level of serum creatinine, withstatistically significant difference from the nephritis rats withplacebo treatment.

3. The Effect of Peptides on the Spleen Indexes TABLE III.3 The effectof peptides on the spleen index(×10⁻³) Groups n spleen index CMS014 103.6 ± 1.3* CMS018 10 3.3 ± 0.8* CMS030 10 3.0 ± 0.5* CMS036 10 3.4 ±0.8* placebo 10 4.8 ± 1.1  hydrocortisone 10 4.5 ± 1.4  normal 9 2.4 ±0.1*Comparing with nephritis rats receiving placebo treatment (placebo), *p< 0.05

The spleens of all induced rats were enlarged compared with normal rats,showing that the induction of nephritis was related to immune response.It was found that the peptides CMS014, CMS018, CMS030, and CMS036 at 50μg/kg/day once per day were able to decrease the spleen index, withstatistically significant difference from the group of nephritis ratsreceiving placebo treatment, p<0.05. This suggested that the peptidesmight exert their corrective effect against nephritis throughimmunosuppression mechanisms.

4. The Effects of Peptides on the Renal Pathological MicroscopicExamination

Comparing with normal rats, the nephritis rats receiving placebotreatment (placebo group) showed signs of fibrous tissue formation inthe glomerular capsule, hyperplasia of the glomerular epithelium,formation of crescents, dilation and congestion of glomerularcapillaries, edema of the proximal tubule epithelium, and cast formationin the distal tubule and collecting duct. These pathological changesconfirmed the successful induction of nephritis. It was observed that inthe group CMS014, there was only one rat showed signs of fibrous tissueformation in the glomerular capsule and hyperplasia of the glomerularepithelium. Other parameters of the same rat, and other rats of the samegroup have essentially the same renal histology as normal rats. In thegroups CMS018, CMS030, and CMS036, all pathological parameters of allrats were normal.

Conclusion

It is concluded that the peptides CMS014, CMS018, CMS030, and CMS036 at50 μg/kg/day, once per day, administered intraperitoneally can havetherapeutic effect on experimental Masugi nephritis rat model.Proteinurea, creatinine excretion, and renal histology of the treatedrats were corrected by these peptides, with statistically significantdifference from the control group receiving placebo treatment. Thesepeptides may act through immunological mechanisms as indicated by theirinfluence on the spleen weight index, but does not exclude thepossibility of their actions via other mechanisms.

Discussion

The peptides CMS014, CMS018, CMS030, and CMS036 maybe useful as a part,or on its own, in nephritis management in human. For example, thepeptides may be used to correct proteinurea or to restore the excretaryfunctions of nephritis patients. The peptides may also be used toprevent further kidney function impairment in nephritis patients. Thepeptides may be used on its own, in combinations of two or morepeptides, or in combination with other pharmaceuticals or foodsupplements as a total course for nephritis management. TABLE III.4Peptides Effective for Nephritis CMS code SEQ ID No. CMS014 7 CMS018 10CMS030 21 CMS036 26References1. SDA(State Drug Administration, P.R. China). The guideline ofpreclinical researches of new drugs. 1994, the 1st edition, Page 962. Xu Shuyun, et al. The methodology of pharmacological experiment, thePeople's Sanitation Publishing Company, Beijing, the 2nd edition, 1991:1071.3. Chen Qi, et al. The methodology of pharmacological researches oftraditional Chinese medicine, the People's Sanitation PublishingCompany, Beijing, the 1st edition. 1993: 390.4. Du Guanhua. The guideline for pharmacological experiment - thediscovery and pharmacological evaluation of new drugs, SciencePublishing Company, Beijing, the 1st edition, 2001: 598.5. Wang Shuxian. Nephrology, the People's Sanitation Publishing Company,Beijing, the 11^(th) edition, 1987: 244.IV. Effect of CMA Peptides on Cancer

In order to find out whether these peptides have possible therapeutic oncancer, we used various standard animal cancer models in this study totest out the biological effects of the above peptides on diseasedanimals.

Materials

1. Experimental Animals

BALB/c mice, C₅₇BL/6 mice, and DBA/2 mice, weighing 18-22 g, from ChinaMedical Science Institute, PR China.

2. Cell Lines

Mouse sarcoma S₁₈₀ cells, B₁₆ cells, and L₁₂₁₀ cells, from CancerResearch Department, China Medical Science Institute.

YAC-1 cells, as gift from Prof Yao Zhi, Tianjin Medical University.

3. Main Drug and Reagent

The peptides used in this study were custom manufactured by AmericanPeptide Company, Inc., USA.

Fetal bovine serum, RPMI-1640 cell culture medium, from Gibco, USA.

MTT, ConA, from Sigma, USA.

Recombinant mouse interferon-γ (rmIFN-γ), from Beijing Biotech Inc., PRChina.

Recombinant human interleukin-2 (rhIL-2), from Shanghai Huaxin BiotechInc., PR China.

Lymphocyte separation solution, from Research Institute of HematologicDisease, National Institute of Medical Science, PR China

Cyclophosphamide, from The 12th pharmaceutical factory of Shanghai, PRChina.

II. Methods

1. Administration of Test Substance

Intraperitoneal injection, once per day. With the exception ofcyclophosphamide group, all groups started treatment 5 days beforetransplanting the cancer cells. The cyclophosphamide groups were treatedon the next day after transplanting the cancer cells. Treatment of allgroups with testing substance lasted for 30 days or until the animaldied unless otherwise specified.

2. The Effect of Peptides on the Growth Rate of Transplanted S₁₈₀Sarcoma Cells in BALB/c Mice, and that on the Immunological Function ofthe Host

BALB/c mice were randomized into peptide group, cyclophosphamide group,rmIFN-γ group, rhIL-2 group, and saline group, 20 animals per group.

Stock S₁₈₀ sarcoma cells were incubated in DMEM/F12 medium supplementedwith 10% fetal bovine serum, 37° C., 5% CO₂ for 72 hrs, then washed 3-4times with Hank's solution at room temperature. Adjust the cellconcentration to 1-2×10⁹ per litre with Hank's solution. 0.2 ml of cellsuspension was implanted to several BALB/c mice through the armpit for10-12 days. The mice were killed by cervical vertebra dislocation.Vigorously growing and non-disrupted tumor masses were harvested andwashed clean with sterile saline. The tissue was dispersed in saline toa homogenous cell suspension, in a ratio of 1 g tissue to 4 ml saline.The sarcoma bearing mice model was prepared with an injection of 0.2 mlcell suspension through the armpit [1]. Administration of test substancetreatment started as described in section Method 1.

2.1 The effect of peptides on the phagocytotic function of mononuclearphagocyte in mice with S₁₈₀ sarcoma [2,3] was analysed by injecting micefrom the tail vein with 0.1 ml/10 g body weight of Indian ink (1:5dilution with normal saline) on the second day after the last testsubstance administration. At one minute and five minutes after theinjection, 20 μl blood was obtained from the eye canthus withheparinised tubing. The blood was mixed with 2 ml 0.1% w/v Na₂CO₃ andthen OD₆₈₀ nm obtained. The outline clear index K was calculated by thefollowing formula:K=(1 g A ₁−1 g A ₂)÷(t2−t1)

-   -   Key:    -   A1: OD₆₈₀ nm at first minute    -   A2: OD₆₈₀ nm at fifth minute    -   t2: 5 minutes    -   t2: 1 minute

After the phagocytosis index study, the mice were killed by cervicalvertebra dislocation. The liver, spleen, and cancer tissue weredissected, blotted dry, and weighed.

The phagocytosis index α was calculated as below:α=(³ √{square root over ( )}K)×(W÷W _(LS))

-   -   key:    -   W: body weight    -   W_(LS): weight of liver plus spleen

2.2 The tumor growth inhibition index was calculated according to thefollowing formula:Tumor growth inhibition index=(mean tumor weight of control group−meantumor weight of treatment group)÷mean tumor weight of control group3. The Effect of Peptides on the Survival of BALB/c Mice withTransplanted Ascitic Fluid Type Liver Cancer H₂₂

BALB/c mice were randomly grouped into peptide group, cyclophosphamidegroup, rmIFN-γ group, rhIL-2 group and saline group, 20 animals pergroup.

Stock H₂₂ cells were incubated in DMEM/F12 medium supplemented with 10%fetal bovine serum, 37° C./5% CO₂ for 72 hrs, then washed 3-4 times withHank's solution at room temperature. Adjust cell concentration to1-2×10⁹ per litre with Hank's solution. 0.2 ml of the cell suspensionwas implanted to the abdominal cavity of several BALB/c mice for 6-8days [1]. The mice were killed by cervical vertebra dislocation. Asciticfluid of the mice was collected asceptically and the cell concentrationadjusted to 1×10⁶ per ml with Hank's solution. 0.2 ml of the cellsuspension was implanted into the abdominal cavity of healthy mice togenerate the H₂₂ carrying mouse model for ascitic fluid type livercancer. Administration of test substance started as described in sectionMethod 1. Survival data of the mice was recorded. If the animal survivedlonger than the experiment, the survival days was recorded as theduration of the experiment. Mean survival day was obtained by theKaplan-meier method in the Survival option of the SPSS software. Thesurvival index was calculated according to the following formula:Survival index=(mean survival days in treatment group−mean survival daysof control group)÷mean survival days of control group×100%4. The Effect of Peptides on the Cellular Immunity of BALB/c Mice withTransplanted Ascitic Fluid Type Liver Cancer H₂₂4.1 Preparation of Spleen Cell Suspension^([1,4])

Healthy BALB/c mice were randomised into peptide group, rmIFN-γ group,rhIL-2 group, and saline group, 15 mice per group, and prepared into H₂₂carrying mice model as described in section Method 3. After implantationof the cancer cells, the test substances were administered for 15 days,the mice were killed by cervical dislocation. The spleen was isolatedaseptically and manually dispersed in cold D-Hank's solution using aninjection needle. The dispersed cell suspension was further sievedthrough a 100-gauge 150 μm diameter stainless steel sieve. Aftercentrifugation at 200 g for 10 minutes, the supernatant was discarded.The cell pellet was re-suspended in 10 volume of Tris-NH₄Cl buffer andthen kept standing still for 10 minutes at room temperature. Thesuspended cells were collected by centrifugation at 150 g for 10minutes. The cells were washed 2-4 times with cold D-Hank's solution byre-suspending and collecting by centrifugation with condition asdescribed above. The washed cells were then diluted to the desired celldensities by RPMI-1640 culture medium, containing 10% fetal bovineserum.

4.2 The Effect of Peptides on the T Lymphocyte Transformation in micewith Ascitic Fluid Type Liver Cancer H₂₂ ^([1,4])

Spleen cells of density 1×10⁶/ml were placed onto a 96 wells cellculture plate, 100 μt/well, three parallel wells each of the assaysample and control sample per mouse. To the assay wells, 100 μl/wellConA of 100 μg/ml in RPMI-1640 was added, and 100 μl/well plainRPMI-1640 was used for the controls. The cells were incubated for 66 hrsat 37° C., 5% CO₂. The cells were then pelleted by centrifuigation at150 g for 10 minutes. The supernatant was collected and stored at −20°C. for cytokines IL-2 and IFN determination.

50 μl/well MTT of 1 mg/ml in RPMI-1640 was added to the cell pellet andthe cells re-suspended by shaking for 2 minutes. The incubation wascontinued for 4 hours. The supernatant was discarded aftercentrifugation at 150 g for 10 minutes. 120 μl 40 mM HCl-2-propanol wasadded to the cell pellet and shaken for 3 minutes. Use an ELISA readerto obtain OD₅₇₀ nm of each well referenced at 630 nm.

Each mouse formed three assay wells and three control wells. TheStimulation Index (SI) of each mouse was obtained by first deriving theaverage OD of the three parallel wells, then dividing the value of theassay wells by the control wells.

4.3 The Effect of Peptides on the NK cell Activity in Mice with AsciticFluid Type Liver Cancer H₂₂ ^([5, 6])

Mice spleen cells were prepared to 4×10⁶/ml as described in section 4.1above. Target cells YAC-1 were brought to log phase and adjusted to1×10⁵/ml. Using a 96 wells cell culture plate, 100 μl mouse spleen cellsand 100 μl culture medium were added to the control well containing onlythe spleen cells; 100 μl target cells and 100 μl culture medium wereadded to the control well containing only target cells; 100 μl mousespleen cells and 100 μl target cells were added to the NK activity assaywell. Three parallel sets of the above were prepared per mouse. Afterthat, the 96 wells cell culture plates were incubated for 4hrs at 37°C., 5% CO₂.

Samples were centrifuged at 150 g for 10 minutes to collect the cells.The supernatant was discarded and 50 μl/well MTT of 1 mg/ml was added.The reaction-mixture was then shaken for 2 minutes, and incubated at 37°C., 5% CO₂ for 4 hours. The supernatant was discarded aftercentrifugation at 150 g for 10 minutes. 120 μl 40 mM HCl-2-propanol wasadded and shaken for 3 minutes. An ELISA reader was used to obtainOD_(570 nm) of each well referenced at 630 nm.

Each mouse has 9 wells: three spleen cells only control, three targetcells only control, and three assay wells with both spleen and targetcells. The NK cell activity index of each mouse was obtained by firstderiving the average OD of the three parallel wells of each combination,then applying this average OD to the following formula:NK cell activity index=[1−(average OD of spleen and target cellwell−average OD of spleen cell only well)÷(average OD of target cellonly well)]×100%5. The Effect of Peptides on the Survival of DBA/2 Mice withTransplanted L₁₂₁₀ Leukemia

DBA/2 mice, 6-8 weeks old, were randomized into peptide group,cyclophosphamide group, rmIFN-γ group, rhIL-2 group, and saline group,20 animals per group.

Stock L₁₂₁₀ cells were incubated in DMEM/F12 medium supplemented with10% fetal bovine serum, 37° C./5% CO₂ for 72 hrs, then washed 3-4 timeswith Hank's solution, and adjusted to 1×10⁵ cells per litre. 0.1 ml ofthe cell suspension was implanted into the abdominal cavity of severalhealthy DBA mice for 6-8 days. The mice were then killed by cervicalvertebra dislocation and their ascitic fluid collected aseptically. Thecell concentration of the collected ascitic fluid was adjusted to 1×10⁵per ml with Hank's solution. 0.1 ml of the cell suspension was inplantedinto each of the testing animal and the survival data of the animalsrecorded. Treatment started in the testing animals as described insection Method 1. Mean survival day was obtained by the Kaplan-meiermethod in the Survival option of the SPSS software. If the animalsurvived longer than the experiment, the survival days was entered asthe duration of the experiment. The survival index was calculatedaccording to the following formula:Survival index=(mean survival days of treatment group−mean survival daysof control group)÷mean survival days of control group6. The Effect of Peptides on the Humoral Immunity of C₅₇BL/6 MiceBearing Transplanted B₁₆ Melanoma, and that on the Metastatic Potentialof the Inoculated Melanoma Cells

C₅₇BL/6 mice, 6-8 weeks old, body weight 18-22 g, were randomized intopeptide group, cyclophosphamide group, rmIFN-γ group, rhIL-2 group, andsaline group, 20 animals per group.

Stock B₁₆ mouse melanoma cells were incubated in DMEM/F12 mediumsupplemented with 10% fetal bovine serum, 37° C./5% CO₂ for 72 hrs, thenwashed 3-4 times with Hank's solution. The cell concentration wasadjusted to 1×10⁵ per ml with Hank's solution and 0.1 ml of the cellsuspension was injected via the tail vein into the testing mice togenerate the B₁₆ melanoma bearing animal model [7,8]. Treatment withtest substance started as described in section Method 1.

6.1 The Effect of Peptides on the Humoral Immunity of C₅₇BL/6 MiceBearing Transplanted B₁₆ Melanoma^([9])

Sheep red blood cells (SRBC) were prepared by collecting blood from thecervical vein and put into a sterile flask with glass beads. The flaskwas shaken for 3 minutes and the blood then mixed with Alsever solution(glucose 2.05 g, NaCl 0.4 g, Na lemonade 0.8 g, adjust to 100 ml withdistilled water) and stored at 4° C. Immediately before use, sampleswere centrifuged at 130 g, 5 minutes to collect the SRBC. The cells werewashed two times by re-suspension and centrifugation in normal saline.Then the cell pellet was collected by centrifugation at 180 g for 10minutes and re-suspended in saline to make the fmal working SRBCsuspension, 2% (v/v).

Complement was prepared by adding 10 volumes of fresh Cavy serum intoone volume centrifuge packed SRBC, and then gently shaked for 30 minutesat 4° C. The SRBC was removed by centrifugation at 200 g for 10 minutes.10 volumes of normal saline were added to obtain the working complementsolution.

On the testing animals, on the 27^(th) date of test substance treatment,0.2 ml of the working SRBC cell suspension was injected into each animalto raise antibody. On the day after the last test substanceadministration, blood was collected from the eye canthus and left atroom temperature for one hour for serum exudation. After centrifugationat 200 g for 10 minutes, the collected serum was diluted by 500 timeswith normal saline.

To 1 ml diluted mouse serum of each mouse, 0.5 ml SRBC suspension wasadded. Ice cold. Then 1 ml working complement solution was added andincubated at 37° C. water bath for 10 minutes. Reactions were terminatedby ice cold. Samples were then centrifuged at 200 g for 10 minutes toobtain the supernatant.

To 1 ml of this supernatant, 3 ml Drabkin solution was added and left atroom temperature for 10 minutes. OD₅₄₀ nm was obtained.

Reference OD₅₄₀ nm was obtained by mixing 0.25 ml SRBC suspension withDrabkin solution to 4 ml and placed at room temperature for 10 minutesbefore OD540 nm was taken.Hemolysis index=(OD_(540 nm) of test sample÷reference OD_(540 nm))×5006.2 After the humoral immunity study, the mice were killed by cervicalvertebra dislocation. Postmortum examination of the animals wasperformed. The pathological changes were recorded, and the numbers ofmelanoma lung metastasis foci were counted.Results

1. The Effect of Peptide on the Cellular Phagocytosis in BALB/c Micewith Transplanted S₁₈₀ Sarcoma (Method 2.1) TABLE IV.1 The effect ofpeptide on the phagocytosis index of BALB/c mice with transplanted S₁₈₀sarcoma Group Dose N Phagocytosis index CMS001 50 μg/kg 20 6.24 ±0.33*{circumflex over ( )} CMS001 5 μg/kg 19 6.67 ± 0.43*{circumflexover ( )} CMS034 5 μg/kg 19 6.20 ± 0.44*{circumflex over ( )} CMS034 0.5μg/kg 20 6.35 ± 1.02* IL-2 3 × 10⁵ IU/kg 19 6.96 ± 1.37* IFN-γ 3 × 10⁵IU/kg 17 5.45 ± 0.71 Cyclo-phos 20 mg/kg 19 5.92 ± 2.47 phamide Saline0.5 ml 19 5.38 ± 0.85*comparing to saline, P < 0.05{circumflex over ( )}comparing to rmIFN-γ, P < 0.05

CMS001 at 50 μg/kg/day and 5 μg/kg/day, and CMS034 at 5 μg/kg/day and0.5 μg/kg/day were found to be able to increase the phagocytosis index,having statistically significant difference from the saline group.

2. The Effect of Peptide on the Growth Rate of Transplanted S180 Sarcomain BALB/c Mice (Method 2.2) TABLE IV.2 The effect of peptide ontransplanted S180 tumor growth Tumor inhibition Group Dose N Tumorweight(g) index (%) CMS010 500 μg/kg 20 0.67 ± 0.35* 48.4 CMS034 0.5μg/kg 20 0.83 ± 0.48* 35.9 CMS035 5 μg/kg 20 0.71 ± 0.37* 44.6 IL-2 3 ×10⁵ IU/kg 20 0.69 ± 0.37* 46.2 IFN-γ 3 × 10⁵ IU/kg 18 0.96 ± 0.45 25.3cyclo-phos 20 mg/kg 20 0.68 ± 0.32* 47.3 phamide Saline 0.5 ml 20 1.29 ±0.50*comparing to saline group, P < 0.05

CMS010 at 500 μg/kg/day, CMS034 at 0.5 μg/day, and CMS035 at 5 μg/kg/daywere found to be able to reduce the growth of the transplanted S₁₈₀sarcoma, having statistically significant difference from the salinegroup (P<0.05).

3. The Effect of Peptide on the Survival of BALB/c Mice withTransplanted Ascitic Fluid Type Liver Caner H₂₂ (Method 3) TABLE IV.3The effect of peptide on the survival index of BALB/c mice withtransplanted ascitic fluid type liver cancer H22 Survival index GroupDose N Survival days (%) CMS008 5 μg/kg 20 50.7 ± 20.9*^(&) 67.8 CMS0115 μg/kg 20 36.4 ± 22.2*^(&) 60.2 CMS024 50 μg/kg 20 36.3 ± 12.7*^(&$)38.4 CMS024 5 μg/kg 19 40.6 ± 14.6*^(&$) 54.8 CMS024 0.5 μg/kg 19 46.4 ±14.8*{circumflex over ( )}^(&$) 76.9 CMS032 0.5 μg/kg 20 42.8 ±12.2*{circumflex over ( )}^(&$) 63.3 rhIL-2 3 × 10⁵ IU/kg 18 13.6 ± 0.5rmIFN-γ 3 × 10⁵ IU/kg 20 27.8 ± 7.5 6.1 Cyclo-phosphamide 20 mg/kg 2024.7 ± 10.2 Saline 0.5 ml 19 26.2 ± 6.8*comparing to saline, P < 0.05{circumflex over ( )}comparing to rmIFN-γ, P < 0.05^(&)comparing to rhIL-2, P < 0.05^($)comparing to cyclophosphamide, P < 0.05

CMS008 at 5 82 g/kg/day, CMS011 at 5 μg/kg/day, CMS024 at 50 μg/kg/day,CMS024 at 0.5 μg/kg/day, and CMS032 at 0.5 μg/kg/day were found to beable to prolong the survival of BALB/c mice with transplanted H₂₂ascitic fluid type liver cancer, having statistically significantdifference from the saline group (P<0.05). It was also observed that inthe group CMS024 0.5 μg/kg/day, more than 30% (n=6) of the mice couldsurvive longer than 90 days (two months after the experiment ended).Postmortum examination of the mice did not show sign of tumorestablishment. CMS024 at 0.51 μg/kg/day therefore may interfere with thegrowth of the transplanted H₂₂ by interfering with its establishment orinducing the complete healing of the established cancer.

4. The Effect of Peptide on T Lymphocyte Transformation in BALB/c Micewith Transplanted Ascitic Fluid Type Liver Cancer H₂₂ (Method 4.2) TABLEIV.4 The effect of peptide on T lymphocyte transformation Group Dose NStimulation index CMS010 500 μg/kg 20 1.45 ± 0.21*^($) CMS019 0.5 μg/kg19 1.50 ± 0.19*^($) CMS024 0.5 μg/kg 19 1.46 ± 0.19*^($) CMS024 5 μg/kg20 1.45 ± 0.21*^($) CMS034 0.5 μg/kg 20 1.37 ± 0.10*^($) CMS035 0.5μg/kg 20 1.40 ± 0.13*^($) CMS035 5 μg/kg 20 1.46 ± 0.16*^($) rhIL-2 3 ×10⁵ IU/kg 19 1.46 ± 0.21* rmIFN-γ 3 × 10⁵ IU/kg 18 1.27 ± 0.14Cyclo-phosphamide 20 mg/kg 19 1.01 ± 0.23* Saline 0.5 ml 20 1.25 ± 0.07*comparing to saline, P < 0.05^($)comparing to cyclophosphamide, P < 0.05

CMS010 at 500 μg/kg/day, CMS019 at 0.5 μg/kg/day, CMS024 at 0.5μg/kg/day and 5 μg/kg/day, CMS034 at 0.5 μg/kg/day and CMS035 at 0.5μg/kg/day and 5 μg/kg/day were found to be able to increase thestimulation index of T-lymphocyte, having statistically significantdifference from the saline group (P<0.05).

5. The Effect of Peptide on the NK Cell Activity in BALB/c Mice withTransplanted Ascitic Fluid Type Liver Cancer H₂₂ (Method 4.3) TABLE IV.5The effect of peptide on the NK cell cytotoxic activity index Group DoseN NK activity index(%) CMS003 500 μg/kg 17 37.9 ± 14.5*{circumflex over( )}^($) CMS014 0.5 μg/kg 17 40.7 ± 19.7*^($) CMS024 0.5 μg/kg 18 39.3 ±18.7*^($) CMS024 5 μg/kg 20 34.9 ± 12.1*{circumflex over ( )}^($) CMS02450 μg/kg 20 43.6 ± 13.9*{circumflex over ( )}^($&) CMS032 5 μg/kg 2052.6 ± 12.5*{circumflex over ( )}^($&) CMS032 50 μg/kg 19 41.0 ±18.7*{circumflex over ( )}^($&) CMS034 50 μg/kg 20 57.3 ±17.9*{circumflex over ( )}^($&) IL-2 3 × 10⁵ IU/kg 19 26.0 ± 9.0 IFN-γ 3× 10⁵ IU/kg 18 20.9 ± 3.3 Cyclo-phos 20 mg/kg 19 16.5 ± 7.2* PhamideSaline 0.5 ml 20 24.0 ± 8.2*as compared with saline, P < 0.05.{circumflex over ( )}as compared with IFN-γ, P < 0.05.^(&)as compared with IL-2, P < 0.05.^($)as compared with cyclophosphamide, P < 0.05

CMS003 at 500 μg/kg/day, CMS014 at 0.5 μg/kg/day, CMS024 at 0.5μg/kg/day, 5 μg/kg/day, and 50 μg/kg/day, CMS032 at 5 μg/kg/day and 50μg/kg/day and CMS034 at 50 μg/kg/day were found to be able to increasethe NK cell cytotoxic activity in the testing animal model, havingstatistically significant difference from the saline group (P<0.05).

6. The Effect of Peptide on the Survival of DBA/2 Mice with TransplantedL₁₂₁₀ Leukemia (Method 5) TABLE IV.6 The effect of peptide on thesurvival index of testing animals Survival Group Dose N Survival daysindex (%) CMS019 0.5 μg/kg 20 21.1 ± 5.8* 26.8 CMS035 0.5 μg/kg 20 29.3± 15.4* 76.1 IL-2 3 × 10⁵ IU/kg 20 32.0 ± 13.7* 92.3 IFN-γ 3 × 10⁵ IU/kg20 15.6 ± 2.2 Cyclophosphamide 20 mg/kg 20 24.0 ± 5.3* 44.2 Saline 0.5ml 21 16.6 ± 5.6*comparing to saline group, P < 0.05

CMS019 at 0.5 μg/kg/day and CMS035 at 0.5 μg/kg/day were found to beable to prolong the survival of DBA/2 mice with transplanted L1210leukemia, having statistically significant difference from the salinegroup (P<0.05).

7. The Effect of Peptide on the Humoral Immunity of C₅₇BL/6 Mice withTransplanted B₁₆ Melanoma (Method 6.1) TABLE IV.7 The effect of peptideon the hemolysis index of C57BL/6 mice with transplanted B16 melanomaGroup Dose N Hemolysis index CMS001 0.5 μg/kg 20 51.0 ± 16.2*^($) CMS0015 μg/kg 20 41.3 ± 17.7*^($) CMS001 50 μg/kg 19 45.0 ± 31.9*^($) CMS001500 μg/kg 20 36.0 ± 10.2*^($) CMS003 0.5 μg/kg 20 61.6 ± 26.9*^($)CMS003 5 μg/kg 20 37.2 ± 15.9*^($) CMS003 50 μg/kg 20 38.7 ± 13.5*^($)CMS003 500 μg/kg 20 35.9 ± 13.0*^($) CMS008 0.5 μg/kg 19 42.7 ±18.4*^($) CMS008 5 μg/kg 20 38.9 ± 12.0*^($) CMS008 50 μg/kg 20 37.1 ±16.7*^($) CMS008 500 μg/kg 20 50.1 ± 17.8*^($) CMS010 0.5 μg/kg 18 34.9± 10.5*^($) CMS010 5 μg/kg 20 51.0 ± 14.6*^($) CMS010 50 μg/kg 20 39.6 ±7.7*^($) CMS010 500 μg/kg 20 50.1 ± 16.7*^($) CMS011 0.5 μg/kg 20 32.0 ±14.7* CMS011 500 μg/kg 20 34.4 ± 19.4* CMS016 500 μg/kg 20 43.3 ± 29.9*CMS019 0.5 μg/kg 20 42.0 ± 12.0*^($) CMS019 5 μg/kg 20 35.4 ± 15.1*^($)CMS019 50 μg/kg 20 28.3 ± 7.6* CMS024 0.5 μg/kg 20 43.0 ± 10.7*^($)CMS024 5 μg/kg 20 42.2 ± 11.8*^($) CMS024 50 μg/kg 20 27.8 ± 9.1* CMS024500 μg/kg 18 30.1 ± 10.0* CMS034 0.5 μg/kg 18 50.8 ± 18.4*^($) CMS034 5μg/kg 19 43.0 ± 11.7*^($) CMS034 50 μg/kg 20 30.2 ± 10.9* CMS035 5 μg/kg20 38.9 ± 21.2*^($) CMS035 50 μg/kg 20 44.7 ± 22.7*^($) CMS035 500 μg/kg19 40.5 ± 25.8* rhIL-2 3 × 10⁵ IU/kg 19 49.3 ± 24.7* rmIFN-γ 3 × 10⁵IU/kg 19 60.5 ± 17.4* Cyclo-phosphamide 20 mg/kg 19 20.7 ± 19.1 Saline0.5 ml 20 19.0 ± 9.1*comparing to saline, P < 0.05{circumflex over ( )}: comparing to rmIFN-γ, P < 0.05&: comparing to rhIL-2, P < 0.05^($)comparing to cyclophosphamide, P < 0.05

CMS001, CMS003, CMS008, CMS010, CMS011, CMS016, CMS019, CMS024, CMS034,and CMS035 were found to be able to enhance the humoral response(increase in hemolysis index) of C₅₇BL/6 mice with transplanted B₁₆melanoma at dosages as shown on Table IV.7, having statisticallysignificant difference from the saline group (P<0.05).

8. The Effect of Peptide on the Survival of Inoculated B16 MelamonaCells in C₅₇BL/6 Mice (Method 6.2)

Postmortum examination of animals after the ending of test substancetreatment did not show any sign of existance of B16 metastatic foci inthe lung of mice treated with CMS008 at 0.5 μg/kg/day, 5 μg/kg/day, and50 μg/kg/day, and CMS016 at 5 μg/kg/day and 500 μg/kg/day.

III. Conclusion

In compliance with the Preclinical New Drug Research Guidelines issuedby Branch of Drug Administration, Department of Health, PR China in1993, the effects of peptide on mice with transplanted cancer cells wasstudied. It was concluded that:

-   1. CMS010, CMS034, and CMS035 at suitable dosage could significantly    inhibit the development of transplanted S180 sarcoma in mice;-   2. CMS001 and CMS034 at suitable dosage could enhance the    phagocytotic immune activities of mice transplanted with S180    sarcoma;-   3. CMS008, CMS011, CMS024, and CMS032 at suitable dosage could    prolong the survival of mice transplanted with ascitic fluid type    liver cancer H22;-   4. CMS010, CMS019, CMS024, CMS034, and CMS035 at suitable dosage    could enhance T lymphocyte transformation in mice transplanted with    ascitic fluid type liver cancer H22;-   5. CMS003, CMS014, CMS024, CMS032, and CMS034 at suitable dosage    could increase the NK cell cytotoxic activity of mice transplanted    with ascitic fluid type liver cancer H22;

6. CMS019 and CMS035 at suitable dosage could prolong the survival ofmice transplanted with L1210 leukemia;

-   7. CMS008 and CMS016 at suitable dosage could inhibit the    development of transplanted B16 melanoma in mice;-   8. CMS001, CMS003, CMS008, CMS010, CMS011, CMS016, CMS019, CMS024,    CMS034, and CMS035 at suitable dosage could enhance the humoral    immune response of mice transplanted with B16 melanoma.    Discussion

CMS001, CMS003, CMS008, CMS010, CMS011, CMS014, CMS016, CMS019, CMS024,CMS032, CMS034, CMS035 may be useful as a part, or on its own, in cancermanagement in human. For example, CMS001, CMS003, CMS008, CMS010,CMS011, CMS014, CMS016, CMS019, CMS024, CMS032, CMS034, and CMS035 maybe used to increase the immunity of cancer patients. CMS008, CMS010,CMS016, CMS034, and CMS035 may be used to interfer with the growth ofcancer cells in patients. CMS008, CMS011, CMS019, CMS024, CMS032, andCMS035 may be used to prolong the life expectancy of cancer patients.The peptides may be used on its own, in combinations of two or morepeptides, or in combination with other pharmaceuticals or foodsupplements as a total course for cancer management. TABLE IV.8 PeptidesEffective for Cancer CMS code SEQ ID No. CMS001 1 CMS003 27 CMS008 3CMS010 4 CMS011 30 CMS014 7 CMS016 9 CMS019 11 CMS024 16 CMS032 22CMS034 24 CMS035 25Reference1. Principles of Pre-clinical Research of New Drugs, People's Republicof China. 1993, 7: 137-1432. Principles of Pre-clinical Research of New Drugs, People's Republicof China. 1993, 7: 128-1293. Yuanpei Zhang, Huaide Su. Pharmacological experiment (secondedition). People's Health Publishing House. 1998, 137-1384. Shuyun Xu, Rulian Bian, Xiu Chen. Methodology of pharmacologicalexperiment. People's Health Publishing House. 1991, 1221-12345. Principles of Pre-clinical Research of New Drugs, People's Republicof China. 1993, 7: 1406. Jinsheng He, Ruizhu Li, Tingyi Zong. The study on MTT reductionmethod of testing NK cell activity. China Immunology Journal. 1996,1(6): 356-3587. Yaoqin Yang, Huchuan Yang, Huihong Tao, et al. The synergic effect ofTween-80 on the antitumor of hyperthermia-Experimental studies of mousemelanoma. Cancer Research on Prevention and Treatment. 1999, 26(4): 8-128. Jian Fu, Jie Zheng, Weigang Fang, et al. Interleukin-12 genetransfection into murine B16 melanoma cells suppresses tumorigenicityand decreases metastatic potential. National Medical Journal of China.1998, 78(8): 627-6299. Qian Wang. Modern medical experiment method. People's HealthPublishing House. 1998, 482-48310. Qichao Pan, Bin Xu. Cancer pharmacology and chemotherapy. Henanmedical university Publishing House. 2000, 66-6911. Yuanpei Zhang, Huaide Su. Pharmacological experiment (secondedition). People's Health Publishing House. 1998, 131V. The Effect on the Body Weight

Healthy rats were feeded with high nutrition diet for 5 weeks, with orwithout simultaneous peptide treatment (intramuscular 300 μg/kg/day).Rats on an ordinary diet with saline injection were used as negativecontrol. After 5 weeks of treatment, injection was stopped and the samediet was maintained for another three weeks. Body weight data wascollected at once per week interval. Behaviour of the rats was alsoobserved. Rats receiving peptide CMS015 were found to have statisticallysignificant lower body weight increase compared with control during thecourse of peptide treatment. This trend of decreased body weightgradually reduced after ceasing the CMS015 treatment. It is concludedthat CMS015 at suitable dosage level can reversibly control obesitydevelopment induced by over-feeding.

Materials

Sprague-Dawley (SD) rats, weighing 145±10 g, were supplied by the Centerof Experimental Animal of Guangzhou University of Traditional ChineseMedicine, PR China (certificate No.: 2000A019). Peptides were customsynthesized (of L-amino acids origin) by American Peptide Company, Inc.,USA, and were diluted to 10 μg/ml in normal saline. The high and normalnutrition diets were prepared in compliance with the guideline forpre-clinical research of anti-obesity drug, issued by SDA (State DrugAdministration), PR China^([1]).

Methods

Healthy rats were randomized into the experiment, positive control, andnegative control groups. 10 rats per group, half male and half female.The experiment group of rats was feeded with high nutrition diet for 5weeks with simultaneous intramuscular injection of 300 μg/kg/daypeptide, once per day. Positive control group received the same highnutrition diet but placebo saline injection, while negative controlgroup, used to proof successful establishment of obesity model, receivednormal nutrition diet and placebo saline injection. After 5 weekstreatment, injection was stopped and the same diet was maintained foranother 3 weeks. The rats were weighed at once per week interval.Behaviour of the rats was also observed.

Statistics

The data were presented as mean±standard deviation. Paired t test orsingle-factor ANOVA was used for inter and intra group comparison.Statistical significance cut-off was at P≦0.05.

Results

1. Effect of Peptide on the Body Weight of SD rat TABLE V.1 Effect ofpeptide on the body weight of SD rats Positive CMS015 Control Group (g)Treatment Group (g) Male n = 5 Female n = 5 Male n = 5 Female n = 5 Pre-145.6 ± 13.6 133.6 ± 4.6 145.6 ± 8.5 129.2 ± 3.3 treatment Week 1 194.4± 14.5 164.4 ± 8.7 183.8 ± 10.6 157.8 ± 8.3 Week 2 239.6 ± 13.4 188.0 ±6.4 220.6 ± 12.2* 176.0 ± 11.2* Week 3 265.0 ± 11.8 208.8 ± 8.2 239.0 ±16.0* 196.0 ± 10.5* Week 4 287.4 ± 17.7 227.2 ± 8.2 258.2 ± 18.1* 212.0± 13.5* Week 5 299.4 ± 21.2 236.6 ± 10.9 268.8 ± 17.7* 221.4 ± 13.2*Week 6 333.4 ± 27.1 249.4 ± 16.3 299.4 ± 21.2* 235.6 ± 16.3 Week 7 349.2± 28.9 261.2 ± 13.4 310.4 ± 25.9* 242.2 ± 18.8* Week 8 374.4 ± 37.2255.6 ± 11.5 337.4 ± 30.6 252.8 ± 22.5In comparison with positive control group: * p < 0.05

At the dose of 300 μg/kg/day, CMS015 was found to be able to limit theweight gain of obesity rats induced by over-feeding, havingstatistically significant difference from the control group (p<0.05).The difference of the treatment and control groups increased with thelength of treatment. Upon termination of peptide CMS015 treatment in thetreatment group, the difference of the treatment and control groupsgradually reduced and became statistically insignificant after threeweeks, showing that the effect of the peptide on SD rat body weight isreversible.

During the whole course of the experiment, it was observed that theappetite and activity of all groups of rats remained normal.

Discussion

It is concluded that CMS015 at suitable dosage level can limit thedevelopment of obesity induced by over-feeding. The peptide may put intohuman use for control of obesity. The peptides may be used on its own,in combinations of two or more peptides, or in combination with otherpharmaceuticals or food supplements as a total course for obesitymanagement.

In the present study, administration by intramuscular injection wastested, but this does not exclude the possible effectiveness of thepeptide if administered via other alternative routes. The peptides maybe administered via intravenous injection, intramuscular injection,intraperitoneal injection, subcutaneous injection, and subcutaneousimplantation, with or without delivery facilitating device such asliposome, sustain release protection etc. The peptide may also beadministered in any form of oral administration like tablet, capsule,suspension, solution etc, in the usual form without modification or inslow release form, or with or without gastro-enteric protection. Thepeptide may further be applied in any form of topic application likeointment, cream, gel etc with or without transdermal facilitatingdevice, or as inhalant of powder, dissolved, or as liposome protectedform. The peptide may also be interpreted into its genetic sequence andcloned into an expression system, on its own or in combination withother peptide sequences, to generate a resulting peptide molecule tomake use of the activity of the peptide as described in this report,with or without purification of the resulting peptide. TABLE V.2Peptides Effective for Obesity CMS code SEQ ID No. CMS015 8References1. SDA, PR China. The guideline for pre-clinical research of new drugs.1993

It is understood that it may be possible to add additional amino acidsto the amino or carboxyl termini of the above disclosed peptides asanother method of practising the present invention. For example, one ortwo amino acids may be added to the disclosed peptide without affectingits biological function. It may also be possible to add three or fouramino acids and still maintain the function of the peptides. These areall referred to as variants of the same peptide. Alternatively, one ortwo amino acids may be deleted from the peptide without affecting itsbiological activity. It may further be possible for three or four aminoacids to be deleted without affecting the biological function of thepeptides. These are referred to as fragments of the instant peptide.Furthermore, derivatives of the peptide such as conservative replacementof one amino acid for another within the same functional class may beused to practise another aspect of the present invention. For example,peptides having non-polar or hydrophobic side chains may be possible tosubstitute one side group for another without reducing biologicalactivity. As a further example, linker/spacer may be inserted into thepeptide to form variants, but the variants still retaining its activemoiety as the original peptide used in this study These are alsoconsidered variants of the peptides. A peptide analogue as used herein,includes peptides that have amino acid molecules which mimic thestructure of the natural amino acid e.g. an analog with a differentbackbone structure, or D-amino acid substitution. As a further example,although the amino acids used for synthesizing the peptides are in theirL optical isomeric form, peptides with one or more of the amino acids inthe sequence substituted with the D-form may have similar biologicalactivities. The term “functional derivative” as used in the claims ismeant to include fragments, variants, analogues or chemical derivativesof the peptide.

As used herein, the term “hybrid peptide” is used to refer to peptidesthat contain additional peptides inserted into the original biologicallyactive peptides having SEQ. ID no. 1-30 or their functional derivatives,but still retain substantially similar activity. The additional peptidesinclude leader peptides that contain, for example, an amino acidsequence that is recognized by one or more prokaryotic or eukaryoticcell as a signal for secretion of the hybrid protein into the exterioror the cell. The secretion may be a direct secretion, or indirectlythrough secretory vesicles.

“Substantially pure peptide” refers to peptides that are at least 10%w/w in purity, more preferably 20%, even more preferably 40% and muchmore preferably 60% and far more preferably larger than 90% pure. In themost preferred embodiment, the purity is larger than 99%. Thesubstantially pure peptide can be used to prepare pharmaceutical andnutritional formulations that may be complex mixtures as describedbelow.

The use of the above-identified peptides in pharmaceutical formulationsmay be employed as possible treatment for immunological disorders ordisease having secondary effect on immunity e.g. cancer or infections orany of the conditions mentioned above. The formulations may contain oneof identified peptides mixed with other active or inactive constituents,including other peptides. E.g. two to several (e.g. 3-5) of the listedpeptides may be added to the same formulation with or without otheringredients. Alternatively, one of the listed peptides may be used toprepare the formulation together with peptides not listed here. They canbe administered in the form of intravenous, intramuscular,intracutaneous, subcutaneous or intradermal. The mode of administrationmay also be intra-arterial injection that leads directly to the organ ofproblem. Other modes of administration are transdermal, inhalation aspowder or spray, and other forms of delivery known by one in the art.The formulation may also be orally taken, and may contain carriers thatcan be used to prevent gastric digestion of the peptide after oralintake or any other carriers known in the art (for transdermal such asliposome).

The pharmaceutical formulation may include any of the knownpharmaceutical carriers. Examples of suitable carriers include any ofthe standard pharmaceutically accepted carrier known to those skilled inthe art. These include but are not limited to, physiological salinesolution, water, emulsions including oil and water mixtures ortriglyceride emulsions, and other types of agents, fillers, coatedtablets and capsules. The appropriate carrier may be selected based onthe mode of administration of the pharmaceutical composition.

The peptides may be administered via intravenous injection,intramuscular injection, intraperitoneal injection, subcutaneousinjection, and subcutaneous implantation. The peptide may also beadministered in any form of oral administration like tablet, capsule,suspension, solution etc, in the usual form without modification or inslow release form, or with or without gastro-enteric protection. Thepeptide may further be applied in any form of topic application likeointment, cream, gel etc with or without transdermal facilitatingdevice. The peptide may also be interpreted into its genetic sequenceand cloned into an expression system, on its own or in combination withother peptide sequences, to generate a resulting peptide molecule tomake use of the activity of the peptide as described in this report.

The dose of each peptide may be 1 ng-10 g per kg body weight. Apreferred dose is 10 ng-10 mg per kg, and more preferably 1 μg-1 mg perkg for an injection mode of administration. However, the effective dosecan be as low as 1 ng per kg body weight, since one or more of thepeptides may operate through receptors that will induce a cascade ofnormal physiological response. Alternatively, one or more of thepeptides can just be an initiator for a whole cascade of reaction. Foran oral intake, the amount may be 1 ng-10 g per day per kg body weight,more preferably 0.1 μg-1 g per day per kg body weight and even morepreferably 1 μg-10 mg per day.

VI. Gene Therapy and Method of Treatment

Gene therapy based on the discovered peptide sequences is performed bydesigning a nucleic acid sequence that code for one of these peptides.The nucleic acid may be synthesized chemically and operably ligated to apromoter, and cloned into an expression vector. The expression vector isthen administered into the human body as the form of gene therapy forexpression in the human cell. The term “genetic vectors” as used hereinincludes these expression vectors. Vectors that can be used for genetherapy includes adeno-associated virus (Mizuno, M. et al. (1998). Jpn JCancer Res 89, 76-80), LNSX vectors (Miller, A. D. et al. (1993) MethodsEnzymol 217, 581-599) and lentivirus (Goldman, M. J. et al. (1997) HumGene Ther 8, 2261-2268).

Other vehicles for peptide delivery include expression vectors encodingthe desired peptide that can be transferred into an organism which canreplicate in the host organism to which it is desired to administer thepeptide without significant detrimental effects on the health of thehost organism. For example, the expression vectors may be transferredinto an organism which is not pathogenic to the host organism to whichit is desired to administer the peptide. In some embodiments theexpression vector produces the desired peptide in a bacterial or fungalorganism which does not have significant detrimental effects on thehealth of the host organism to which the peptide is to be administered.For example, the expression vector encoding the desired peptide may bean expression vector which produces the desired peptide in an organismsuch as lactic acid bacteria, E. coli, or yeast. In one embodiment, theexpression vector produces the desired peptide in a microbe normallyfound in the mammalian gut or a microbe tolerated by the mammaliandigestive tract. Some of the microbial species in which the desiredpeptide can be expressed include, but are not limited to, Lactobacillusspecies, such as L. acidophilus, L. amylovorus, L. casei, L. crispatus,L. gallinarum, L. gasseri, L. johnsonii, L. paracasei, L. plantarum, L.reuteri, L. rhamnosus or others; Bifidobacterium species, such as B.adolescentis, B. animalus, B. bifidum, B. breve, B. infantis, B. lactis,B. iongum or others; Enterococcus faecalis or Ent. facium;Sporolactobacillus inulinus; Bacillus subtilis or Bacillus cereus;Escherichia coli; Propionibacterium freudenreichii; or Saccharomycescerevisiae or Saccharomyces boulardii

Nucleic acid sequences that encode the peptides of the presentinvention, chemically synthesized or produced by other means, includingbut not limited to the reverse transcription of mRNA to produce cDNAmolecules, are incorporated into expression vectors for gene transferinto the desired organisms by methods of genetic engineering familiar tothose of skill in the art. The expression vectors may be DNA vectors orRNA vectors. For example, the expression vectors may be based on plasmidor viral genetic elements. The expression vectors may be vectors whichreplicate extra-chromosomally or vectors which integrate into thechromosome.

The expression vectors comprise a promoter operably linked to a nucleicacid encoding a peptide of the present invention. The promoter may be aregulatable promoter, such as an inducible promoter, or a constitutivepromoter. In some embodiments, the promoter may be selected to provide adesired level of peptide expression. In addition, if desired, theexpression vectors may comprise other sequences to promote theproduction, presentation and/or secretion of peptides. In someembodiments a nucleic acid encoding a peptide of the present inventionis operably linked to a nucleic acid sequence which directs thesecretion of the peptide. For example, the nucleic acid encoding thepeptide of the present invention may be operably linked to a nucleicacid encoding a signal peptide.

In some embodiments, the expression vectors which are engineered toencode the peptides of the present invention may be expression vectorswhich are adapted for expressing the peptide of the present invention ina bacterial species that makes up the normal gut flora of mammals, suchas Lactobacillus species and Bacillus subtilis Examples of suchexpression vectors can be found in U.S. Pat. No. 6,100,388, to Casas,and U.S. Pat. No. 5,728,571, to Bellini, respectively. These documentsare hereby expressly incorporated by reference in their entireties. Itwill be appreciated that any expression vector which facilitates theexpression of a peptide of the present invention in an organism which isnot detrimental to the health of the host organism to which the peptideis to be administered may be used.

In some embodiments, the expression vectors which are engineered toencode the peptides of the present invention may be expression vectorswhich are adapted for expressing the peptide of the present invention ina yeast species that is well tolerated by the mammaliam gut, such asSaccharomyces cerevisiae; or, preferably, Saccharomyces boulardii, whichcan colonize the human gut and is used to treat certain forms ofdiarrhea. Yeast expression vectors can be used that constitutivelyexpress heterologous proteins and peptides, are highly stable, thus arewell transmitted to progeny cells during mitosis and meiosis and maycomprise coding sequence for a signal peptide or peptides that directhigh levels of recombinant protein secretion. An example of such a yeastvector is given in U.S. Pat. No. 6,391,585, to Jang et al., which ishereby expressly incorporated by reference in its entirety.

The expression vectors encoding the peptides of the present inventionmay be introduced into the organism in which it is intended to expressthe peptides through techniques known in the art. These techniquesinclude traditional methods of transforming bacteria, yeast, or othermicrobes, through the use of chemically competent bacterial cells,electroporation or lithium acetate transformation (for yeast), forexample, as well as recent advances in the transformation of bacterialspecies recalcitrant to these procedures. In some embodiments, theexpression vectors are introduced into lactic acid bacteria known to berecalcitrant to transformation using the method disclosed by Leer et al.(WO 95/35389), the disclosure of which is incorporated herein byreference in its entirety. The introduced sequences may be incorporatedinto microbial chromosomal DNA or may remain as extrachromosomal DNAelements.

This genetically engineered microbe containing the expression vector canthen be inoculated into the alimentary canal, vagina, trachea etc. toachieve sustained immuno-therapy. In some embodiments, the organismsexpressing the peptides of the present invention are ingested in aninactive form or, preferably, in live form. In the gut thesemicroorganisms produce said peptides, release them into the lumen bysecretion or by lysis of the microorganism or otherwise present thepeptides to the host, whereby the peptides produce their intended effectupon the host organism. In other embodiments, peptides are presented tothe host at the mucous membrane of the nasal passages, vagina or thesmall intestine.

Another method of the treatment is the use of liposomes as a means fordelivering the specific nucleic acid to the cells in the human body. Thenucleic acid (such as an expression vector containing a nucleic sequencethat encodes peptides of sequence ID No.1 to ID No.30) is delivered inan environment that encourages cellular uptake and chromosomalincorporation as described in Gao, X. and Huang, L. (1995) Gene Ther 2,710-722 and U.S. Pat. No. 6,207,456. Alternatively, the peptide itselfcan be encapsulated in the liposome and delivered directly, using amethod described in U.S. Pat. No. 6,245,427. All the scientificpublications and patents indicated above are incorporated herein byreference.

The nucleic acid sequences useful for the above-mentioned gene therapyand method of treatment include sequences that code for these peptidesand functional derivatives thereof. Any one of the numerous nucleic acidsequences may be used to code for these peptides and their derivativesbased on the degenerate codon system.

The following references are incorporated herein by reference in theirentirety.

-   1. Principles of Pre-clinical Research of New Drugs, People's    Republic of China. 1993, 7:134-135-   2. Shuyun Xu, Rulian Bian, Xiu Chen. Methodology of pharmacological    experiment. People's Health Publishing House. 1991, 1221-1234-   3. Principle of new drug research in pre-clinic issued by Ministry    of Health, People's Republic of China. 1993, 7:140-   4. Jinsheng He, Ruizhu Li, Tingyi Zong. The study on MTT reduction    method of testing NK cell activity. China Immunology Journal. 1996,    1(6): 356-358-   5. Qian Wang. Modem medical experiment method. People's Health    Publishing House. 1998, 482-483-   6. Principle of new drug research in pre-clinic issued by Ministry    of Health, People's Republic of China. 1993, 7: 141-   7. Principle of new drug research in pre-clinic issued by Ministry    of Health, People's Republic of China. 1993,7: 132-133-   8. Principle of new drug research in pre-clinic issued by Ministry    of Health, People's Republic of China. 1993, 7: 128-129

9. Yuanpei Zhang, Huaide Su. Phamalogical experiment (second edition).People's Health Publishing House. 1998, 137-138

-   10. Jiatai Li, clinical pharmacology(second edition). People's    Health Publishing House. 1998, 1338-1339.

EXAMPLE 1 Delivery of Peptides through Genetically EngineeredLactobacillus bacterial Species

The following is provided as one exemplary method to deliver peptides ofthis invention to a host as described above. A DNA sequence that encodesone of the peptides listed in table A above is synthesized by chemicalmeans and this DNA sequence is inserted into an expression vector usingstandard techniques of genetic engineering familiar to those skilled inthe art. The expression vector selected contains a constitutive promoterfunctional in Lactobacilli, a multiple cloning site for the introductionof DNA sequences in a specific 5′ to 3′ orientation as well as aselectable marker gene that confers resistance to an antibiotic (to aidin cloning procedures) and may comprise other sequences to assist in theproduction and/or secretion of the peptides, such as signal peptidesequences. An example of such a vector is provided by U.S. Pat. No.5,592,908, to Pavla, which is hereby incorporated by reference in itsentirety. Briefly, this patent discusses several known promoters thatfunction in Lactobacillus species, as well as a method for discoveringnovel promoters in said bacteria, any of which may be operably linked toa nucleic acid encoding a peptide of the present invention to expressthe peptide in Lactobacilli. A nucleic acid encoding a signal peptide,such as peptides comprising of 16 to 35 mostly hydrophobic amino acidsthat are active in Lactobacillus lactis described in U.S. Pat. No.5,529,908, cited above, is interposed between the promoter and thenucleic acid encoding the peptide of the present invention such that thethe nucleic acid encoding the signal peptide is in frame with thenucleic acid encoding the peptide of the present invention.

In addition to the coding sequence of the peptide, the DNA sequencesynthesized may comprise sequences to aid in the ligation and cloning ofsaid DNA into the expression vector. For example, restriction enzymerecognition sites that correspond to ones found in the multiple cloningsite of the vector can be incorporated into the synthesized DNA at the5′ and 3′ ends of the sequence, so that the sequence can be cloned inproper orientation within the vector. Both the vector and thesynthesized DNA are digested with the particular restriction enzymes,then purified. Ligation reactions with the vector and the synthesizedDNA are followed by transformation into a suitable strain of E. Coli.The transformed bacteria are plated on media containing the antibioticto which the vector confers resistance. A colony of transformed bacteriais selected for growth cultures and plasmid preparation procedures; thepresence of the synthesized DNA in the correct orientation is confirmed.

This expression vector is then transformed into a bacterial host cell ofa Lactobacillus species, such as L. acidophilus. Transformed cells areselected for by virtue of the selectable marker found within the vectorsequence and the secretion of the peptide may be verified by performinga western blot, performing gel electrophoresis of peptides present inthe growth medium or other standard techniques. A transformed colony ofbacteria is chosen and used to prepare large-scale cultures of thegenetically engineered bacteria. A culture of the genetically engineeredbacteria expressing the desired peptide is grown up and at least aportion thereof is administered to the alimentary canal, vagina, tracheaor other area of the host organism in which the bacteria are able toreplicate. If desired, the bacterial cultures can be treated in avariety of ways to produce a supplement for enteric consumption by thehost. These treatments include lyophilization or other methods ofpreserving the bacteria, in addition to combining the bacteria withcarrier agents, such as solutions, solvents, dispersion media, delayagents, emulsions and the like. The use of these agents to preparesupplements is well known in the art. For example, the bacteria can beused to make cultured milk products or other foodstuffs for humanconsumption, such that the organism expressing the peptide colonizes thegut of the host organism. A number of different methods forincorporating specific strains of lactic acid bacteria into foodstuffssuch as yogurt, kimchee, cheese and butter are disclosed in U.S. Pat.No. 6,036,952, to Oh, which is hereby incorporated by reference in itsentirety. Upon consuming the bacteria through one of any number ofroutes, the engineered organisms can colonize the gut and allow thepresentation and/or absorption of the peptides of this invention via themucosal layer of the gut.

EXAMPLE 2 Delivery of Peptides through a Genetically Engineered form ofBacillus subtilis

The following is provided as another exemplary method to deliverpeptides of this invention to a host as described above. A DNA sequencethat encodes one of the peptides listed in table A above is synthesizedby chemical means and this DNA sequence is inserted into an expressionvector via techniques of genetic engineering, all techniques being knownin the art. The expression vector selected comprises a shuttle vector,such as pTZ18R (Pharmacia, Piscataway, N.J.), capable of beingpropagated in both E. Coli and B. Subtilis and containing an antibioticresistance gene for selecting colonies of transformed bacteria. Thisvector can contain a constitutive promoter active in B. subtilis, suchas a promoter derived from the Sac B gene of B. subtilis as well as anucleotide sequence encoding a signal peptide active in B. subtilis thatdirects efficient export of expressed heterologous proteins from thebacterial cell. An example of such a vector is disclosed in U.S. Pat.No. 6,268,169, to Fahnestock, the disclosure of which is incorporatedherein by reference in its entirety. Briefly, as detailed above, the DNAencoding a peptide of this invention will be synthesized withrestriction enzymes sites and/or other sequences to facilitate cloningof the DNA through techniques familiar to those with skill in the art.After transformation into E. Coli., plating, selection and propagationof the plasmid to create a plasmid stock, the plasmid is then betransformed into B. subtilis and transformants are selected by virtue ofresistance to an antibiotic in the plating media.

Peptide production in and secretion from the genetically engineered B.subtilis is verified using techniques well known to those with skill inthe art, such as radiolabeling of peptides for autoradiographicdetection after SDS-PAGE anaylsis or Western blotting.

A culture of genetically engineered bacteria is grown up and at least aportion thereof is administered to the alimentary canal, vagina, tracheaor other area of the host organism in which the bacteria are able toreplicate.

EXAMPLE 3 Delivery of Peptides through Genetically EngineeredSaccharomyces Yeast Species

The following is provided as another exemplary method to deliverpeptides of this invention to a host as described above. A DNA sequencethat encodes one of the peptides listed in table A above is synthesizedby chemical means and this DNA sequence is inserted into an expressionvector via techniques of genetic engineering, all techniques being knownin the art. The expression vector selected comprises a stably maintainedyeast protein expression vector, comprising a constitutive yeastpromoter such as pADH1, sites for replication of the vector in bothyeast and E. Coli, a gene or genes that confer prototrophy to anauxotrophic yeast mutant for selection purposes, a multiple cloning site(MCS) and, if desired, sequences that code for a signal peptide. Vectorssuch as this are commercially available and well known in the art or canbe readily constructed using standard techniques After insertion of thesynthesized DNA into the yeast vector, transformation into E. Coli,plating of transformed E. Coli onto selective media, selection of atransformed bacterial colony and preparation of plasmid DNA from agrowth culture of bacteria from said colony, the vector is transformedinto Saccharomyces cerevisiae via well-known techniques such as lithiumacetate transformation or electroporation. The strain of Saccharomycescerevisiae selected for transformation is a mutant auxotrophic strainthat will require a gene on the plasmid in order to grow on minimalmedia plates. Transformed yeast colonies are isolated by plating theyeast on growth media lacking the gene provided on the vector. Onlythose yeast that have received the vector and its selective gene and areexpressing that gene product will be able to grow into colonies on theminimal media. Verification of peptide secretion can be obtained byperforming a Western blot, performing gel electrophoresis of peptidespresent in the growth medium or other standard techniques.

A transformed colony of yeast is chosen and used to prepare large scalecultures. A culture of the genetically engineered yeast expressing thedesired peptide is grown up and at least a portion thereof isadministered to the alimentary canal, vagina, trachea or other area ofthe host organism in which the bacteria are able to replicate. Ifdesired, the yeast cultures can be treated in a variety of ways toproduce a supplement for enteric consumption by the host. Thesetreatments include lyophilization or other methods of preserving yeast,in addition to combining the bacteria with carrier agents, such assolutions, solvents, dispersion media, delay agents, emulsions and thelike. The use of these agents to prepare supplements is well known inthe art. In another embodiment, the transformed yeast are used in thecreation of food products, such as fermented milk products like yogurtand kefir, by techniques known to those skilled in the art. As with livelactic acid bacterial cultures in these foodstuffs, the transformedyeast colonize the gut at least transiently and serve to presentpeptides to the host via the gut lumen.

1-29. (canceled)
 30. An isolated and substantially pure peptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs. 2, 3, 5-15, 17-30, and a functional derivative thereof. 31.An isolated and substantially pure peptide according to claim 30 whereinsaid peptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs. 2, 3, 5-15,17-30.
 32. An isolated andsubstantially pure peptide according to claim 31 wherein said peptideconsists essentially of an amino acid sequence selected from the groupconsisting of SEQ ID NOs. 2, 3, 5-15,17-30.
 33. An isolated andsubstantially pure peptide according to claim 32 wherein said peptideconsists of an amino acid sequence selected from the group consisting ofSEQ ID NOs. 2, 3, 5-15,17-30.
 34. An isolated and substantially purepeptide according to claim 33 wherein said peptide modulates a conditionselected from the group consisting of immune activity, hepatitisinfection, hepatitis B infection, the extent of nephritis and the growthof a cancer.
 35. A pharmaceutical composition comprising an isolated andsubstantially pure peptide comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs. 2, 3, 5-15, 17-30, and afunctional derivative thereof.
 36. A pharmaceutical compositionaccording to claim 35 wherein said peptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOs. 2, 3,5-15,17-30.
 37. A pharmaceutical composition according to claim 36wherein said peptide consists essentially of an amino acid sequenceselected from the group consisting of SEQ ID NOs. 2, 3, 5-15,17-30. 38.A pharmaceutical composition according to claim 37 wherein said peptideconsists of an amino acid sequence selected from the group consisting ofSEQ ID NOs. 2, 3, 5-15,17-30.
 39. A pharmaceutical composition accordingto claim 38 wherein said peptide modulates a condition selected from thegroup consisting immune activity, hepatitis infection, hepatitis Binfection, the extent of nephritis and the growth of a cancer.
 40. Agenetic vector comprising a nucleotide sequence encoding a peptide, saidpeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs. 1-30, or a functional derivative thereof. 41.A genetic vector according to claim 40 wherein said peptide comprises anamino acid sequence selected from the group consisting of SEQ ID NOs.1-30.
 42. A genetic vector according to claim 41 wherein said peptideconsists essentially of an amino acid sequence selected from the groupconsisting of SEQ ID NOs. 1-30.
 43. A genetic vector according to claim42 wherein said peptide consists of an amino acid sequence selected fromthe group consisting of SEQ ID NOs. 1-30.
 44. A genetic vector accordingto claim 43 wherein said peptide modulates a condition selected from thegroup consisting of immune activity, hepatitis infection, hepatitis Binfection, the extent of nephritis and the growth of a cancer.
 45. Amicro-organism having a genetic composition comprising a nucleotidesequence encoding a peptide, said peptide comprising a biologicallyactive peptide or a functional derivative thereof, said biologicallyactive peptide having an amino acid sequence selected from the groupconsisting of SEQ ID NOs. 1-30.
 46. A method of making a pharmaceuticalcomposition comprising providing an isolated and substantially purepeptide and admixing said peptide with a pharmaceutically acceptablecarrier, wherein said peptide comprises an amino acid sequence selectedfrom the group consisting SEQ ID NO. 1-30, and a functional derivativethereof.
 47. A method of making a pharmaceutical composition accordingto claim 46 wherein said peptide comprises an amino acid sequenceselected from the group consisting SEQ ID NO. 1-30.
 48. A method ofmaking a pharmaceutical composition according to claim 47 wherein saidpeptide consists essentially of an amino acid sequence selected from thegroup consisting SEQ ID NO. 1-30.
 49. A method of making apharmaceutical composition according to claim 48 wherein said peptideconsists of an amino acid sequence selected from the group consistingSEQ ID NO. 1-30.
 50. A method of treating a condition of a mammalcomprising administering a pharmaceutically effective dose of anisolated and substantially pure peptide, said peptide comprising anamino acid sequence selected from the group consisting of SEQ ID NOs. 2,3, 5-15, 17-30, and a functional derivative thereof, wherein said mammalis a human or a non-human animal body.
 51. A method of treating acondition of a mammal according to claim 50 wherein said peptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs. 2, 3, 5-15,17-30.
 52. A method of treating a condition of amammal according to claim 51 wherein said peptide consists essentiallyof an amino acid sequence selected from the group consisting of SEQ IDNOs. 2, 3, 5-15, 17-30.
 53. A method of treating a condition of a mammalaccording to claim 52 wherein said peptide consists of an amino acidsequence selected from the group consisting of SEQ ID NOs. 2, 3,5-15,17-30.
 54. A method of treating a condition of a mammal accordingto claim 53 wherein said condition comprises modulation of the immuneactivity and the growth of cancer.
 55. A method of treating a conditionof a mammal according to claim 54 wherein said modulation of the immuneactivity comprises selecting an immune activity modulating peptide orpeptide-containing composition and administering an effective amount ofsaid peptide or peptide-containing composition to said animal, whereinsaid peptide consists of SEQ ID NOs. 2, 3, 5-15, 17-30, wherein saidamount is effective to modulate immune activity of said animal.
 56. Amethod of treating a condition of a mammal according to claim 54 whereinsaid modulation of the growth of cancer comprises selecting a cancercell proliferation inhibiting peptide or peptide-containing compositionand administering an effective amount of said peptide orpeptide-containing composition to said cells, wherein said peptideconsists of SEQ ID NOs. 2, 3, 5-15, 17-30, wherein said amount iseffective to suppress proliferation or inhibit growth of cancer cells.